Preparation for involving originating from bone marrow pluripotent stem cell into peripheral blood flow

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry and represents a preparation for involving a mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow, which is introduced into the blood vessel or muscle and which contains any of components: (a) protein HMGB1; (b) HMGB1 protein-secreting cell; (c) a vector, into which HMGB1 protein-coding DNA is inserted; (d) protein HMGB2; (e) HMGB2 protein-secreting cell; (f) a vector, into which HMGB2 protein-coding DNA is inserted; (g) protein HMGB3; (h) HMGB3 protein-secreting cell; and (i) a vector, into which HMGB3 protein-coding DNA is inserted.

EFFECT: elaboration of the preparation for involving the mesenchymal stem cell of the bone marrow into peripheral blood from the bone marrow.

3 cl, 6 ex, 1 tbl, 14 dwg

Technical area

The present invention relates to funds that involve originating from bone marrow pluripotent stem cells in the peripheral blood.

The level of technology

In recent years, it has been revealed that during repair of damaged tissues involves various stem cells, and is currently developing new regenerative medicines that induce the regeneration of functional tissues by attracting a large number of stem cells in the damaged area. To implement these new regenerative medicines, (i) stem cells that may be involved in the damaged area must bein vivoin large quantities; and (ii) shall be allocated/identified the factors that attract stem cells in the damaged area.

Examples of stem cells that may be involved in the damaged area, include tissue stem cells that are present in the damaged area or in adjacent tissues, and originating from bone marrow stem cells present in peripheral blood. In recent years, it has been described that originate from the bone marrow cells are involved in many types of regeneration of damaged tissue, but the mechanism of attraction originating from ostego brain cells in the area of the damage is unknown. Originating from bone marrow cells, as used herein, differ from hematopoietic stem cells that have the potential for differentiation into blood cells (leukocytes and erythrocytes), and include stem cells, which are cells called mesenchymal stem cells from bone marrow or cell groups of the precursors of tissue present in the bone marrow. Mesenchymal stem cells from bone marrow are undifferentiated stem cells with the potential for differentiation into osteoblasts, adipocytes and chondrocytes, and, in addition, they can differentiate into other mesenchymal cells such as fibroblasts, muscle cells, stromal cells and cells of tendons. Recently it has been shown that mesenchymal stem cells from bone marrow differentiate into nerve cells and, in addition, epithelial cells (such as skin keratinocytes and vascular endothelial cells (non-patent document 9). Precursor cells of the tissue is defined as undifferentiated cells that have a unidirectional potential for differentiation into specific tissues/cells, distinct from tissues/cells of the circulatory system, and include undifferentiated cells with the potential for differentiation in mesenchymal tissue, epithelial the fabric, nervous tissue, parenchymatous organs and the vascular endothelium, as mentioned above.

HMGB1 (High Mobility Group Box 1: protein 1 group with high mobility) is a protein with a molecular weight of about 25,000, which exists in almost all types of cellsin vivo. According to recent reports, is known for the following features:

1) HMGB1 regulates gene expression by intracellular binding to DNA to control chromatin structure (non-patent document 1);

2) HMGB1 is secreted from monocytes or macrophages present in inflammatory tissues, under the action of inflammatory cytokines TNF-α, IL-1 and LPS, and extracellular binds to RAGE (receptor for advanced glycation products) (non-patent document 2)inducyruya strong inflammatory reaction (non-patent document 3);

3) HMGB1 is released from induced hypoperfusion of necrotic cells in the surrounding tissue (non-patent document 4);

4) HMGB1 is associated with progression of inflammation in patients with septicemia, severe infectious disease (non-patent document 5);

5) administration of HMGB1 in the area of infarction in the model of myocardial infarction provides division/proliferation of stem cells present in the myocardium, and thus regeneration and functional recovery of the myocardium (patent document 1);

6) the introduction of HMGB1 fashion is gnome animal hypoperfusion liver failure before induction hypoperfusion States softens the degree of disturbance of the liver (non-patent document 6);

7) the introduction of HMGB1 in the area of damage in a model of muscle injury sends simultaneously injected precursor cells of blood vessels in the damaged area, and, thus, stimulates the regeneration of muscle tissue (non-patent document 7); and

8) HMGB1 induces the formation of neurites in nerve cells (non-patent document 8). However, in previous messages not shown that originate from bone marrow stem cells, in particular mesenchymal stem cells that can differentiate into osteoblasts, chondrocytes, adipocytes, etc. are involved in the damaged tissue.

Traditionally believed that the Central nerve cells in the brain and spinal cord cannot regenerate after injury. However, recently became aware of the existence of neural stem cells, and it has become possible induction of these cells. Also identified the niche of neural stem cells in the normal nervous system. Thus, the restoration of the damaged Central neurons, which have long thought impossible, is now considered feasible. In the present study, associated with neuronal regeneration in the brain and spinal cord, degenerative diseases, etc. are expanded.

The main causes of damage to tissue (cells) of the brain are traumati the mini brain injury and ischemic brain disease. Other causes may be damaged due to surgery of the brain, such as the removal of a brain tumor. In particular, the complete removal of neuroglioma developed from the cells of the parenchyma of the brain, is difficult, and there is no other solution but to dwell on partial removal to avoid damage to motor and speech functions. Moreover, malignant neuroglioma has a poor prognosis, and none of the treatments actively investigated in recent years, from chemotherapy and radiation therapy prior to immunotherapy/gene therapy, had no satisfactory effects. Thus, the ideal method of treatment may be a treatment that can remove as many tumor cells as possible and to repair damaged brain function as a consequence of the deletion.

The documents of the prior art

Patent documents

Patent document 1: Publication of patent applications of Japanese Kohyo No. (JP-A) 2005-537253 (pending publication of the national phase in Japan, the corresponding non-Japanese international publication)

Non-patent documents

Non-patent document 1: Bustin et al., Mol. Cell Biol., 19: 5237-5246, 1999.

Non-patent document 2: Hori et al., J. Biol. Chem., 270, 25752-25761, 1995.

Non-patent document 3: Wang et al., Science, 285: 248-251, 1999.

Non-patent document 4: Mulle et al., EMBO J, 20: 4337-4340, 2001.

Non-patent document 5: Wang et al., Science, 285: 248-251, 1999.

Non-patent document 6: Germani et al., J. Leukoc. Biol., Jan; 81(1): 41-5, 2007.

Non-patent document 7: Palumbo et al., J. Cell Biol., 164: 441-449, 2004.

Non-patent document 8: Merenmies et al., J. Biol. Chem., 266: 16722-16729, 1991.

Non-patent document 9: Y. Wu et al., Stem. cells, 25: 2648-2659, 2007.

Description of the invention

Objectives of the invention

It is known that mesenchymal stem cells can differentiate into bone, cartilage and adipose tissue, are among the stem cells in the bone marrow. In recent years, it has been revealed that there are pluripotent stem cells that differentiate into epithelial cells and nerve cells.

Meanwhile, methods of treatment of refractory ulcers of the skin include the treatment of transplantation of the skin. The study authors present invention showed that the skin regenerates through the reconstruction of the epidermis, dermis, hair follicles (tissue that forms hair), and the like, originating from bone marrow cells in the transplanted skin after the treatment of ulcers. Thus, it is expected that can be created in a simple and efficient way of taking such populations of cells with the capacity for repair of tissues from the bone marrow. To date, however, this method is still not developed.

Thus, the task is altoadige of the invention is the provision of methods of involving a large number originating from bone marrow pluripotent stem cells in the peripheral blood.

Means for solving problems

There is a possibility that in the process of engraftment of the transplanted skin on biological tissue in the skin graft involves originating from bone marrow cells of the tissue that are not leather, and, thus, participate in the regeneration of skin tissue. This suggests a potential mechanism involving such pluripotent originate from bone marrow cells in peripheral blood. The present invention provides for the involvement of a large number originating from bone marrow pluripotent stem cells in the peripheral blood by intravenous administration of an extract of skin tissue or inductor originating from bone marrow pluripotent stem cells. Specifically, using the present invention for the first time in the world, discovered that

(1) originating from the bone marrow pluripotent stem cells can be induced in peripheral blood by intravenous tissue extract, obtained from a selection of skin;

(2) a substance selected fragments of the skin, which is responsible for the ongoing involvement of the bone marrow pluripotent stem cells in the peripheral blood, is an HMGB1; and

(3) HMGB1, which has activity involving originate from the bone marrow of pluripotent stolovitch in the peripheral blood, can be easily cleaned from cultured cells.

Based on the discoveries described above, the present invention relates to the following inventions:

[1] a tool for engaging the bone marrow cells in the peripheral blood from the bone marrow, which is injected into a blood vessel or muscle and that includes any of the following components:

(a) protein HMGB1;

(b) a cell that secretes a protein HMGB1;

(c) vector, which is embedded in the DNA encoding a protein HMGB1;

(d) protein HMGB2;

(e) a cell that secretes a protein HMGB2;

(f) vector, which is embedded in the DNA encoding a protein HMGB2;

(g) HMGB3 protein;

(h) a cell that secretes HMGB3 protein; and

(i) vector, which is embedded in the DNA encoding a protein HMGB3;

[2] the tool for engaging the bone marrow cells in the peripheral blood from the bone marrow, which is produced by a process comprising a stage dive cells or tissue in a solvent and injected into a blood vessel or muscle;

[3] the tool for engaging the bone marrow cells in the peripheral blood from the bone marrow, which is injected into a blood vessel or muscle and which contains a heparin-binding fraction, obtained by a process comprising a stage

(a) immersing the cells or tissue in a solvent;

(b) contacting the immobilized heparin with the extract obtained in stage (a); and

(c) elwi the Finance heparin-binding fractions with immobilized heparin;

[4] a way to assess whether the factor involves the cell bone marrow into the peripheral blood from the bone marrow, in the extract of cells or tissues, and when the activity involvement of bone marrow cells in the peripheral blood from the bone marrow at the stage (b) exceeds the activity of the control, determine what factor is contained in the extract of cells or tissue, where the method includes the stages shown below:

(a) obtaining an extract of cells; and

(b) measuring the activity involvement of bone marrow cells in the peripheral blood from the bone marrow in the extract obtained in stage (a);

[5] the method of screening of the extract of cells or tissues containing the factor that involves cell bone marrow into the peripheral blood from the bone marrow, which includes stage

(a) evaluation of multiple extracts of the method according to [4], does the extract factor, which involves cell bone marrow into the peripheral blood; and

(b) selecting extract, which according to the assessment in stage (a) contains a factor that involves the cell bone marrow into the peripheral blood from the bone marrow;

[6] the method for the identification of factors involving cell bone marrow into the peripheral blood from the bone marrow, which includes a step of purification of the factor involving cell bone marrow into the peripheral is kind of the blood from the bone marrow, from the extract, which is defined as containing the factor involving cell bone marrow into the peripheral blood from the bone marrow, the method according to [4] or [5], using the active involvement of bone marrow cells in the peripheral blood from the bone marrow as an indicator;

[7] set to engage the bone marrow cells in the peripheral blood from the bone marrow containing composition, subject to introduction into a blood vessel or muscle, and which contains any of the substances:

(a) protein HMGB1;

(b) a cell that secretes a protein HMGB1;

(c) vector, which is embedded in the DNA encoding a protein HMGB1;

(d) protein HMGB2;

(e) a cell that secretes a protein HMGB2;

(f) vector, which is embedded in the DNA encoding a protein HMGB2;

(g) HMGB3 protein;

(h) a cell that secretes HMGB3 protein; and

(i) vector, which is embedded in the DNA encoding a protein HMGB3;

[8] set to engage the bone marrow cells in the peripheral blood from the bone marrow, containing an extract of cells or tissues, subject to introduction into a blood vessel or muscle and which is produced by the method including a stage dive cells or tissue in a solvent;

[9] a kit for the involvement of bone marrow cells in the peripheral blood from the bone marrow, containing the heparin-binding fraction, subject to introduction into a blood vessel or muscle kotoruyu get way includes stage

(a) immersing the cells or tissue in a solvent;

(b) contacting the immobilized heparin with the extract obtained in stage (a); and

(c) elution of heparin-binding fractions with immobilized heparin;

[10] the method of involvement of the bone marrow cells in the peripheral blood from the bone marrow, including the stage of introduction into a blood vessel or muscle of any of the following substances:

(a) protein HMGB1;

(b) a cell that secretes a protein HMGB1;

(c) vector, which is embedded in the DNA encoding a protein HMGB1;

(d) protein HMGB2;

(e) a cell that secretes a protein HMGB2;

(f) vector, which is embedded in the DNA encoding a protein HMGB2;

(g) HMGB3 protein;

(h) a cell that secretes HMGB3 protein; and

(i) vector, which is embedded in the DNA encoding a protein HMGB3;

[11] the method of involvement of the bone marrow cells in the peripheral blood from the bone marrow, which includes a step of introducing into a blood vessel or muscle extracts of cells or tissues obtained by a process comprising a stage dive cells or tissue in a solvent;

[12] the method of involvement of the bone marrow cells in the peripheral blood from the bone marrow, including the stage of introduction into a blood vessel or muscle heparin-binding fractions, obtained by a process comprising a stage

(a) immersing the cells or tissue to dissolve the ü;

(b) contacting the immobilized heparin with the extract obtained in stage (a); and

(c) elution of heparin-binding fractions with immobilized heparin;

[13] use any of the following substances (a)-(i) in the manufacture of means for involvement of bone marrow cells in the peripheral blood from the bone marrow, which is injected into a blood vessel or muscle:

(a) protein HMGB1;

(b) a cell that secretes a protein HMGB1;

(c) vector, which is embedded in the DNA encoding a protein HMGB1;

(d) protein HMGB2;

(e) a cell that secretes a protein HMGB2;

(f) vector, which is embedded in the DNA encoding a protein HMGB2;

(g) HMGB3 protein;

(h) a cell that secretes HMGB3 protein; and

(i) vector, which is embedded in the DNA encoding a protein HMGB3;

[14] the use of an extract of cells or tissues obtained by a process comprising a stage dive cells or tissue in a solvent, to obtain the means for engaging the bone marrow cells in the peripheral blood from the bone marrow, which is subject to introduction into a blood vessel or muscle;

[15] the use of a heparin-binding fraction, obtained by a process comprising a stage

(a) immersing the cells or tissue in a solvent;

(b) contacting the immobilized heparin with the extract obtained in stage (a); and

(c) elution of heparin-binding fractions with them is mobilizovannogo heparin;

to obtain the involvement of bone marrow cells in the peripheral blood from the bone marrow, which is subject to introduction into a blood vessel or muscle;

[16] any substance of the following:

(a) protein HMGB1;

(b) a cell that secretes a protein HMGB1;

(c) vector, which is embedded in the DNA encoding a protein HMGB1;

(d) protein HMGB2;

(e) a cell that secretes a protein HMGB2;

(f) vector, which is embedded in the DNA encoding a protein HMGB2;

(g) HMGB3 protein;

(h) a cell that secretes HMGB3 protein; and

(i) vector, which is embedded in the DNA encoding a protein HMGB3;

which is used in the method involving the bone marrow cells in the peripheral blood from the bone marrow, which is subject to introduction into a blood vessel or muscle;

[17] extract of cells or tissue obtained by the method including a stage dive cells or tissue in a solvent, which is used in the method involving the bone marrow cells in the peripheral blood from the bone marrow and which is subject to introduction into a blood vessel or muscle; and

[18] the heparin-binding fraction is obtained by a process comprising a stage

(a) immersing the cells or tissue in a solvent;

(b) contacting the immobilized heparin with the extract obtained in stage (a); and

(c) elution of heparin-binding fractions with immobilized heparin;/p>

which is used in the method involving the bone marrow cells in the peripheral blood from the bone marrow and which is subject to introduction into a blood vessel or muscle.

Brief description of figures

Figure 1 presents a diagram that shows expressing vector HMGB1.

Figure 2 presents a diagram that shows the introduction of an extract of the skin (SE) mouse through the tail vein, followed by collecting peripheral blood.

Figure 3 presents the scheme of fractionation, flow cytometry mononuclear fraction of peripheral blood cells of a mouse, a fluorescently labeled antibody against mouse PDGFRα and antibody against CD44 mice 12 hours after administration of an extract of the skin (SE). The top three graphs correspond to the group injection of PBS (n=3) as a negative control, and the bottom three graphs correspond to the group administration of an extract of the skin (SE) (n=3). The vertical axis indicates the level of expression of CD44 and the horizontal axis indicates the level of expression of PDGFRα. The area enclosed in the frame, corresponds to the population of double-positive for CD44 and PDGFRα-cells. The population was increased in the group administration of an extract of the skin (SE) compared to group the introduction of PBS.

Figure 4 presents the scheme of injection of HMGB1 mouse through the tail vein with subsequent capture of the peripheral blood.

Figure 5 presents a diagram which shows the fractionation flow cytometry mononuclear fraction of peripheral blood cells, fluorescently labeled antibody against mouse PDGFRα and antibody against CD44 mice 12 hours after administration of HMGB1. The left diagram corresponds to the mice, which were injected in PBS as a negative control, and the right graph corresponds to the mice that were injected HMGB1. The vertical axis indicates the level of expression of CD44 and the horizontal axis indicates the level of expression of PDGFRα. The area enclosed in the frame, corresponds to the population of double-positive for CD44 and PDGFRα-cells. In mice which were injected HMGB1, the population was increased compared with mice that were administered PBS.

Figure 6 presents a set of photographs showing the detection of Western blot testing of the HMGB family in the extract of the skin of a newborn mouse.

Figure 7 presents a set of photos of the results of Western blotting for collection of purified recombinant fused protein tag Flag-HMGB expressed in HEK293 cells.

On Fig presents a set of graphs showing the activity of recombinant HMGB1/HMGB2/HMGB3 in relation to the migration of bone marrow mesenchymal stem cells using camera Boyden. All recombinant proteins showed higher activity against migration compared with control groups.

Figure 9 presents a set of graphs showing the result of treatment in the model treatment of skin Jaz is in mice using proteins of the HMGB family. All of HMGB1, HMGB2 and HMGB3 showed significant effects in the reduction of the area of ulcers compared with control groups.

Figure 10 presents a photograph showing the activity of HMGB1 on human and extract human skin in relation to the induction of migration originating from human bone marrow mesenchymal stem cells, confirmed using camera Boyden.

Figure 11 presents a set of photographs showing the activity of activators, purified on a column of heparin, from extracts of the heart, brain and skin of the mouse in relation to the induction of mesenchymal stem cells from bone marrow confirmed using camera Boyden.

On Fig presents a set of photographs showing the activity of the extract of cultivated cell lines HEK293 and HeLa extract in relation to the induction of migration of mesenchymal stem cells from human bone marrow confirmed using camera Boyden. Both cultured cell lines showed chemotactic activity towards mesenchymal stem cells human bone marrow.

On figa presents a picture showing a mouse, fixed to a stereotactic device for brain slit of the head in the midline with a scalpel, followed by trephination with use the of borax. On FIGU presents a picture showing the brain, to which is applied a negative pressure using a syringe for sucking part of the brain tissue. On figs presents a photograph of a mouse after injection of 5 µl of purified on a column of heparin fractions of the extract of the skin, dissolved in fibrin adhesive composition (fibrinogen), and the subsequent injection of 5 μl of the composition of fibrin glue (thrombin). On fig.13D and 13E includes photographs of models of brain damage, obtained after 2 weeks of treatment. Higher accumulation of positive GFP cells were observed in the treatment group using purified on a column of heparin fractions of the extract of the skin, E, compared with control, on D. On fig.13F and 13G presents pictures models of brain damage, obtained 6 weeks after treatment. Higher accumulation of GFP-positive cells were observed in the treatment group using purified on a column of heparin fractions of the extract of the skin, G, compared with control, at F.

On figa is a diagram of the flow cytometry, which shows the presence of cells with CD44 and PDGFRα. The introduction of HMGB1 were increased populations as double positive for PDGFRα and CD44 cells and PDGFRα positive, negative CD44 cells in accessories the historical blood. On figv and 14C presents the results of the comparison between groups the introduction of PBS and HMGB1 in relation to the presence of double positive for PDGFRα and CD44 cells, positive for PDGFRα and negative for CD44 cells in the peripheral blood, respectively. Both cell populations were at a statistically significant level increased in the group the introduction of HMGB1.

Ways of carrying out the invention

The present invention relates to pharmaceutical means for engaging bone marrow cells in the peripheral blood from the bone marrow, including one of the following ingredients (a)-(i)subject to introduction into the peripheral blood or muscle:

(a) protein HMGB1;

(b) a cell secreting protein HMGB1;

(c) vector, which is embedded in DNA, which encodes the protein HMGB1;

(d) protein HMGB2;

(e) a cell secreting a protein HMGB2;

(f) vector, which is embedded in DNA, which encodes a protein HMGB2;

(g) HMGB3 protein;

(h) a cell secreting HMGB3 protein; and

(i) vector, which is embedded in DNA, which encodes a protein HMGB3.

With the introduction of the above-described pharmaceutical drugs in a blood vessel or muscle stem cells of bone marrow are involved in peripheral blood, and, thus, it is possible to stimulate the regeneration of damaged tissue. In addition to applying the above pharmaceutical in image quality is as inducer/promoter functional tissue regeneration, expected to its use as a so-called preventive medicines, which prevents damage to the functions of the tissue/organ, caused by the decrease in the number of tissue stem cells, or as a medicine against aging, which slows the progression of age-related changes.

Alternate this treatment can be done by introduction of the above pharmaceutical agents, collection and concentration of pluripotent stem cells, are involved in the peripheral blood from the body, and the introduction of cells in the damaged area. Conventional therapy using mesenchymal stem cells bone marrow is invasive as cells taken from bone marrow, located deep in the body. Meanwhile, with the use of pharmaceuticals of the present invention, mesenchymal stem cells from bone marrow can be collected from peripheral blood in a less invasive way and used for transplantation of cells or similar.

The present invention relates to pharmaceutical means for engaging bone marrow cells in the peripheral blood from the bone marrow, which contain extracts of cells or tissues by the introduction into a blood vessel or muscle, and to the E. receiving means, includes stage dive cells or tissues in a solvent.

Cells or tissue to be immersed in the solvent is not specifically limited, but include, for example, originating from tissue cells, cells, cell lines derived from originating from tissue cells (including, but not limited to, for example, HeLa and HEK293), the selected cell, the unselected cells (e.g. cells in the selected tissues and cells, transfetsirovannyh DNA that encodes a protein HMGB1, HMGB2, or HMGB3. As described above, the tissue can be any tissue. For example, such fabrics include, but are not limited to, the living tissues of the skin or tissue, obtained internal biopsy (surgery) (such as the brain, lung, heart, liver, stomach, intestine, pancreas, kidney, bladder, spleen, uterus, testes and blood).

Examples of the above solvent include, but are not limited to, saline, PBS (phosphate-salt buffer) and TBS (buffered saline Tris). Moreover, the time of immersion of the cells or tissue in the solvent should have a duration which is necessary and sufficient for the induction of cell necrosis, i.e. from 1 hour to 48 hours (for example, from 6 to 48 hours, and preferably from 12 to 24 hours, but it is not limited to this. Thus, the "stage dive cleto is in the solvent" can be paraphrased as "stage dive cells in the solvent for a period of time, necessary and sufficient for the induction of necrosis" or "stage ensure cell death". Moreover, examples of temperature for the immersion of the cells or tissue in a solvent include, but are not limited to, from 4°C to 25°C (for example, from 4°C to 8°C), and preferably 4°C. in Addition, examples of the pH to dive cells or tissue in a solvent include, but are not limited to, pH 7 to 8, preferably pH 7.5. Examples of the buffer include, but are not limited to, phosphate-saline buffer at a concentration of from 10 mm to 50 mm, preferably from 10 to 20 mm.

Moreover, in the present invention, after immersion of the cells or tissues in the solvent may be removed from the solvent containing them. The method of extraction of cells or tissues of the solvent is not specifically limited, provided that the method is well known to specialists in this field. For example, cells or tissues can be removed from the solvent by centrifugation at gravitational acceleration from 10 to 100,000 G G (for example, 440 G) at 4°C to 25°C (for example, 4°C) followed by separation of the supernatant, however, the removal method is not limited to this method. The supernatant can be used as an extract of cells or tissues.

Extracts of cells or tissues according to the present invention, obtained by means includes a stage dive cells or tissues in the solvent, R is t, for example, but not limited to, extract the skin and extract mononuclear cells of peripheral blood (extract of peripheral blood).

Extract of peripheral blood is obtained in the following way: after blood collection syringe or similar, cells frozen in a freezer or in liquid nitrogen, dry ice, or similar methods, and then thawed at a temperature of 0°C or more. Then to remove insoluble cellular components of the sample is centrifuged, for example, when the force of gravity from 10 to 100000 G (for example, when 440 G) and 4°C to 25°C (for example, at 4°C) and the supernatant collected. Insoluble cellular components can be removed from the solvent by the method described above. However, the ways to remove insoluble cellular components is not limited to the above example. The resulting supernatant can be used as an extract of cells or tissues. Alternative instead of centrifugation insoluble cellular components can be removed by filtration through a nitrocellulose filter with 0.45 μm micropore, or other Alternative, collected peripheral blood can be allowed to stand for three to 48 hours at 4°C for induction of cell necrosis. Such treatment of peripheral blood cells can be released intracellular components. Then, removal of the of astorias insoluble cellular components, the sample is centrifuged at a force of gravity from 10 to 100000 G (for example, when 440 G)and the resulting supernatant collected. Insoluble cellular components can be removed from the solvent by the method described above, but is not limited to them. The resulting supernatant can be used as an extract of cells or tissues. Alternative instead of centrifugation insoluble cellular components can be removed by filtration through a nitrocellulose filter with 0.45 μm micropore or other

Meanwhile extracts from mononuclear cells of peripheral blood is obtained in the following way: spend the capture of whole peripheral blood using a syringe or similar thereto, and then the entire sample was diluted up to 4 ml by PBS. After adding 3 ml of Ficoll-Paque Plus (GE) in a centrifuge tube of diluted blood is layered on the Ficoll layer. The test tube is centrifuged at 400 G and 18°C for 40 minutes. The average layer containing mononuclear cells, transferred into a fresh centrifuge tube and add 45 ml of PBS. The sample is centrifuged at 800 G and 18°C for five minutes and the resulting supernatant removed. To the cells again, add 45 ml of PBS and the sample centrifuged at 800 G and 18°C for five minutes. The obtained supernatant removed. To the precipitated cells, add 200 ál of PBS and suspended. The cell suspension allow toatl at -80°C in a freezer for 30 minutes. The frozen suspension is removed from the freezer and thawed on ice. Processing by freezing-thawing is repeated three times. The sample was then centrifuged at 800 G and 4°C for 15 minutes and the supernatant collected. Alternative instead of freezing cells sample cell can be allowed to stand at 4°C for three to 48 hours for the induction of cell necrosis. Through this processing, it is possible to release intracellular components. Alternative cells can be disrupted by sonication under cooling on ice. Through this processing, it is possible to release intracellular components. In any case, after the release of intracellular components of the cell sample is centrifuged at a force of gravity from 440 to 1000000 G, preferably from 20000 to 100000 g of the Obtained supernatant collected as the extract of the cells. Alternative instead of centrifugation insoluble components can be removed by filtration through a filter of nitrocellulose or cellulose acetate with micro pores of 0.45 μm or the like, the Obtained filtrate is used as an extract of cells.

In addition, the present invention relates to pharmaceutical means, subject to introduction into a blood vessel or muscle, to apply for involvement of bone marrow cells in the peripheral blood from the bone marrow, including heparin-binding fraction, produced by the method which comprises the following stages:

(a) immersing the cells or tissue in a solvent;

(b) contacting the extract obtained in stage (a)with immobilized heparin; and

(c) elution of the heparin-binding fraction (also it may be called the purified heparin fraction or purified on a column of heparin fraction) with immobilized heparin.

"Immobilized heparin" refers to the heparin covalently linked to an insoluble carrier. Examples of the insoluble carrier include, but are not limited to, pellet sepharose (such as sepharose 4B, sepharose 6B, etc.: GE Healthcare). In the present invention it is also possible to use commercially available immobilized heparin (column Hitrap Heparin HP: GE Healthcare).

Examples of conditions for contacting an extract of cells or tissues with immobilized heparin include, but are not limited to, a pH of about 7 to 8 (preferably pH 7.5) and the salt concentration from 0 to 200 mm, preferably from about 100 to 200 mm. The time during which the extract is contacted with immobilized heparin, is not specifically limited, however, the contact preferably continues for 5 minutes or more for sufficient adsorption of heparin-binding fractions on immobilized heparin. Examples of temperatures include, but are not on reicived them from 4 to 8°C, preferably 4°C. in Addition, examples of the conditions of elution of heparin-binding fractions adsorbed on immobilized heparin include, but are not limited to, a pH of about 7 to 8 and a salt concentration from 200 to 1000 mm (preferably about 1000 mm).

When injected into a blood vessel or muscle pharmaceuticals containing the extract or fraction described above involve stem cells from the bone marrow into the peripheral blood circulation and can stimulate the regeneration of damaged tissues. Moreover, it is assumed that the above-described pharmaceutical agents can be used not only as inductors/promoters for functional tissue regeneration, but also as a so-called preventive medicines for the prevention of functional disorders of tissues/organs, caused by a decrease in the number of tissue stem cells, or medicines anti-aging to slow the progression of aging-related changes.

Alternate this treatment can be done by introduction of the above pharmaceutical agents, collection and concentration of pluripotent stem cells, are involved in the peripheral blood from the body and then injecting the cells in the damaged area. Accepted what I therapy using mesenchymal stem cells bone marrow is invasive, because cells are collected from bone marrow, located deep in the body. Meanwhile, with the use of pharmaceuticals of the present invention, mesenchymal stem cells from bone marrow can be collected from peripheral blood in a less invasive way and used for transplantation of cells or similar.

The present invention relates to kits for the involvement of bone marrow cells in the peripheral blood from the bone marrow, which consist of songs that are subject to introduction into a blood vessel or muscle, and which contain any of the following materials:

(a) protein HMGB1;

(b) a cell that secretes a protein HMGB1;

(c) vector, which is embedded in the DNA encoding a protein HMGB1;

(d) protein HMGB2;

(e) a cell that secretes a protein HMGB2;

(f) vector, which is embedded in the DNA encoding a protein HMGB2;

(g) HMGB3 protein;

(h) a cell that secretes HMGB3 protein; and

(i) vector, which is embedded in the DNA encoding a protein HMGB3.

Moreover, the present invention relates to kits for the involvement of bone marrow cells in the peripheral blood from the bone marrow, which contain extracts of cells or tissues produced by the method including a stage dive cells or tissues in a solvent, and which are subject to introduction into a blood vessel or muscle.

Also us is Aasee the invention relates to kits for the involvement of bone marrow cells in the peripheral blood from the bone marrow, which contain heparin-binding fraction, subject to introduction into a blood vessel or muscle, and receive, the method including stage

(a) immersing the cells or tissue in a solvent;

(b) contacting the immobilized heparin with the extract obtained in stage (a); and

(c) elution of heparin-binding fractions with immobilized heparin.

The above sets for involvement of bone marrow cells in the peripheral blood, characterized by the fact that the introduction into a blood vessel or muscle involves stem cells from the bone marrow into the peripheral blood stream.

Examples of the above kits include kits for stimulation of tissue regeneration, including (1) the above extract or above faction or similar dissolved in fibrinogen, and (2) thrombin; or alternative sets to stimulate tissue regeneration containing (1) the above extract or above faction or similar, (2) fibrinogen and (3) thrombin. In the present invention can be used commercially available fibrinogen and thrombin. Their examples include, but are not limited to, fibrinogen HT-Wf (Benesis-Mitsubishi Pharma), Beriplast (ZLB Behring), Tisseel (Baxter), Bolheal (Kaketsuken) and TachoComb (ZLB Behring).

Originating from bone marrow cells, which are involved in the injured tissue, differentiate into various the types of cells, involved in functional regeneration of damaged tissue and maintaining/enhancing functions. In the present invention examples of damaged tissue include, but are not limited to, tissue, damaged various physiological conditions, injuries, burns, inflammation, autoimmunity, genetic disorders, etc. that cause the state of ischemia/hypoperfusion/hypoxia. Also damaged tissue includes the necrotic tissue.

Fabric of the present invention is not specifically limited, provided that they can differentiate cells in the bone marrow. Their examples can be all types of tissues in a living body, such as skin tissue, bone tissue, cartilage tissue, muscle tissue, adipose tissue, heart tissue, nervous tissue, lung tissue, tissue of the gastrointestinal tract, the liver/biliary/pancreatic and organs of the urogenital system. Moreover, using the above-described promoters of tissue regeneration are possible treatments for the induction of functional tissue regeneration not only skin disorders such as intractable skin ulcer, skin wounds, bullet and alopecia, but also tissue damage, such as damage due to a heart attack brain, myocardial infarction, bone fracture, heart attack, lung, ulcers as is the RCU and enteritis. The types of animals that may be the introduction of the above-described tissue regeneration promoters include, but are not limited to, human and non-human animals, such as humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters and Guinea pigs.

The bone marrow cells of the present invention are cells other than hematopoietic stem cells, or cells derived from them, such as leukocytes, erythrocytes and platelets, and they include stem cells, which correspond to the cells, up to the present time called mesenchymal stem cells bone marrow stromal pluripotent stem cells in the bone marrow or pluripotent stem cells in the bone marrow, and populations of progenitor cells present in the bone marrow. The bone marrow cells of the present invention can be distinguished from bone marrow extracts (extracts cells of bone marrow) or the collection of peripheral blood. Hematopoietic stem cells are neprekrawajuwimsja, while cells in the bone marrow of the present invention is obtained as a fixed cells by cell culture fraction of mononuclear blood cells derived from bone marrow extracts (extracts cells of bone marrow) or collection perifericos the th blood. Moreover, the bone marrow cells of the present invention include mesenchymal stem cells, and they have the potential to differentiation, preferably, in the osteoblasts (the induction of differentiation can be identified by identifying calcification), chondrocytes (which can be identified by positive staining with alcian blue, positive safranin O, or similar), adipocytes (which can be identified by positive staining with Sudan III), and other mesenchymal cells such as fibroblasts, smooth muscle cells, stromal cells and cells of the tendon; and, in addition, nerve cells, epithelial cells (for example, epidermal keratinocytes and epithelial intestinal cells Express a family of cytokeratins), and vascular endothelial cells. However, the cells, which can occur differentiation, not limited to the above cells, and they also include cells that have the potential for differentiation into cells of parenchymatous organs, such as liver, kidney and pancreas.

In the present invention originating from bone marrow mesenchymal stem cells, pluripotent stromal stem cells from bone marrow or pluripotent stem cells in the bone marrow belong to the cell is m, existing in the bone marrow, which are directly derived from bone marrow or indirectly derived from other tissues (blood, skin, adipose tissue and other tissues)and can be cultivated/they can proliferate as attached cells in culture Cup (made of plastic or glass). These cells are characterized by the potential for differentiation in mesenchymal tissue (mesenchymal stem cells), such as bone, cartilage and adipose tissue, or skeletal muscle, cardiac muscle, in addition, nervous tissue, epithelial tissue (pluripotent stem cells), and you can get them from the collection of blood, bone marrow, peripheral blood or mesenchymal tissues, such as adipose tissue, epithelial tissue, such as skin, nerve tissue such as the brain. Originating from bone marrow mesenchymal stem cells originating from bone marrow pluripotent stem cells or pluripotent stem cells in the bone marrow are also characterized by the presence of the potential for differentiation in epithelial tissue, such as keratinocytes, which form the skin, or nerve tissue, which form the brain, through the introduction of cells attached to the culture plate, in the area of damage to a living organism.

Mesenchymal STV is gross bone marrow cells, pluripotent stromal stem cells from bone marrow or pluripotent stem cells in the bone marrow of the present invention are multipotential stem cells, and they are able to differentiate preferably in osteoblasts (induction of differentiation can be identified by identifying calcification), chondrocytes (which can be identified by positive staining with alcian blue, positive safranin O, or similar), adipocytes (which can be identified by positive staining with Sudan III and so on), and other mesenchymal cells such as fibroblasts, smooth muscle cells, stromal cells and cells of the tendon, nerve cells, liver cells, epidermal cells, hair follicle cells (which Express a family of cytokeratins, family keratin hair or similar), epithelial cells (e.g., epidermal keratinocytes and epithelial intestinal cells Express a family of cytokeratins or similar) and endothelial cells; and, in addition, preferably in cells of the parenchymatous organs, such as liver, kidney and pancreas. However, the differentiated cells are not limited by the above cells.

Moreover, mesenchymal stem cells KOs is tion of the brain, pluripotent stromal stem cells from bone marrow or pluripotent stem cells from human bone marrow can be illustrated by, but not limited to, cells that are directly obtained by collecting bone marrow (extracts of cells of bone marrow, peripheral blood or adipose tissue, or get as attached cells by culturing the selected fraction of mononuclear cells. Markers of mesenchymal stem cells bone marrow stromal pluripotent stem cells in the bone marrow or pluripotent stem cells human bone marrow may represent, for example, but not limited to, some or all of the following markers: Lin-negative, CD45-negative, and CD44-positive.

Moreover, mesenchymal stem cells bone marrow stromal pluripotent stem cells in the bone marrow or pluripotent stem cells in the bone marrow of the mouse can be illustrated by, but not limited to, cells that are directly produced by the methods described in the "Examples"section. Markers of mesenchymal stem cells bone marrow stromal pluripotent stem cells in the bone marrow or pluripotent stem cells in the bone marrow of the mouse can represent, e.g. the measures but not limited to, some or all of the following markers: CD44-positive, PDGFRα-positive, PDGFRβ-positive, CD45-negative, Lin-negative, Sca-1-positive and c-kit-negative.

Precursor cells of the tissue is defined as undifferentiated cells that have a unidirectional potential for differentiation into specific tissue cells other than the circulatory system, and include undifferentiated cells with the potential for differentiation in mesenchymal tissue, epithelial tissue, nervous tissue, parenchymatous organs and the vascular endothelium, as mentioned above.

For pharmaceuticals according to the present invention there are no particular restrictions on components other than extract, heparin-binding fraction, and at least one of ingredients (a)to(i)above, provided that the component is not inhibited attraction of bone marrow cells and the stimulation of tissue regeneration. For example, in addition to the extract, heparin-binding fraction, and at least one of the substances (a)to(i)above, the pharmaceutical agents of the present invention may contain molecules (molecular group), associated with the strengthening of the functions of HMGB1, HMGB2, or HMGB3 for the induction of functional tissue regeneration; molecules (molecular group), which inhibit who have unexpected actions HMGB1, HMGB2, or HMGB3; factors that regulate proliferation and differentiation of bone marrow cells; and other factors that enhance/maintain these factors or cellular functions.

The types of animals that serve as the source of the extract, heparin-binding fractions, HMGB1, HMGB2, or HMGB3 for pharmaceuticals according to the present invention include human and non-human animals, examples of which can be humans, mice, rats, monkeys, pigs, dogs, rabbits, hamsters and Guinea pigs, however, preferably the type of animal is the same as the animal, which intend to enter the above extract or similar.

Protein HMGB1 in the pharmaceuticals of the present invention can be illustrated by, but not limited to, a protein containing the amino acid sequence of SEQ ID NO: 1, 3 or 5. Proteins HMGB1 according to the present invention can also include proteins that are functionally equivalent to the protein containing the amino acid sequence of SEQ ID NO: 1, 3 or 5. Examples of such proteins include 1) the selected proteins that contain the amino acid sequence with one or more amino acid substitutions, deletions, insertions and/or insertions in the amino acid sequence of SEQ ID NO: 1, 3 or 5 and which is functionally equivalent to the be the ku, containing the amino acid sequence of SEQ ID NO: 1, 3 or 5; and 2) selected proteins that are encoded by DNA which hybridize under strict conditions with a DNA containing the nucleotide sequence of SEQ ID NO: 2, 4 or 6 and which is functionally equivalent to a protein containing the amino acid sequence of SEQ ID NO: 1, 3 or 5.

Protein HMGB2 in the pharmaceuticals of the present invention can be illustrated by, but not limited to, a protein containing the amino acid sequence of SEQ ID NO: 7, 9 or 11. Protein HMGB2 of the present invention can also include proteins that are functionally equivalent to the protein containing the amino acid sequence of SEQ ID NO: 7, 9 or 11. Examples of such proteins include 1) the selected proteins that contain the amino acid sequence with one or more amino acid substitutions, deletions, insertions and/or insertions in the amino acid sequence of SEQ ID NO: 7, 9 or 11, and which is functionally equivalent to a protein containing the amino acid sequence of SEQ ID NO: 7, 9 or 11; and 2) selected proteins that are encoded by DNA which hybridize under strict conditions with a DNA containing the nucleotide sequence of SEQ ID NO: 8, 10 or 12, and which is functionally equivalent to a protein containing the amino acid sequence of SEQ ID NO: 7, 9 or 11.

Selected proteins that are functionally equivalent to the protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 may be homologues or paralogue protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. Specialists in this field can identify proteins that are functionally equivale is tiny squirrel containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, by known methods (supplementary volume "Jikken Igaku (Experimental Medicine), Idenshi Kougaku Handbook (Genetic Engineering Handbook)", pp. 246-251, published by YodoshaCo., Ltd., 1991).

Examples of proteins that are functionally equivalent to a protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 include proteins having the activity of inducing originating from bone marrow cells.

Proteins that contain the amino acid sequence with one or more amino acid substitutions, deletions, insertions and/or insertions in the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 and which is functionally equivalent to a protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, include naturally occurring proteins. Typically, eukaryotic genes have polymorphism, as is known genes for interferons and related. Changes in nucleotide sequence, called polymorphism may lead to one or more amino acid substitutions, deletions, insertions and/or inserts. Naturally occurring proteins, such as proteins containing the amino acid sequence with one or more amino acid substitutions, deletions, insertions and/or insertions in the amino acid sequence of SEQ ID NO: 1,3, 5, 7, 9, 11, 13 or 15 and which is functionally equivalent to a protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 included in the protein HMGB1, HMGB2, or HMGB3 of the present invention.

The present invention also includes artificially produced mutant proteins provided that they are functionally equivalent to the protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. Known methods which lead to random mutations of this nucleotide sequence, include the replacement(s) pair(s) of the grounds by processing DNA nitrous acid (Hirose, S.et al., Proc. Natl. Acad. Sci. USA., 79: 7258-7260, 1982). This method provides the random introduction of replace(replacement) pair(s) of bases in a particular segment by processing nitrous acid is desirable for mutations segment. Alternative technologies for site-directed mutations of the target include a method of duplex gap (Kramer, W. and Fritz H.J., Methods in Enzymol., 154: 350-367, 1987), etc. Closed double-stranded vector, in which clone the gene to be embedding mutations, clone and share on a separate circuit. These separate circuit hybridized with a synthetic oligonucleotide with a mutation in the area of the target. Originating from vector complementary single strand of DNA, linearized by the restriction enzyme, are annealed with cycle the single-stranded vector and the gap between the oligonucleotide and the complete vector using a DNA polymerase, which is then converted into a complete double-stranded vector legirovaniem.

The number of amino acids, subject to modification, as a rule, is within 50, preferably in the range of 30, more preferably within 5 amino acids (for example, one amino acid).

When the amino acid artificially replace, replacement of an amino acid having similar properties, leads to the preservation of the activity of the parent protein. Proteins of the present invention include proteins, resulting from conservative substitutions in the above replacement of amino acids(amino acids) and which is functionally equivalent to a protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. A conservative substitution is considered important in the case of replacement of amino acids(amino acids) domains that are important for activity of the protein. Such conservative substitution of amino acids(amino acids) are well known to specialists in this field.

Examples of groups of amino acids suitable for conservative substitutions include basic amino acids (such as lysine, arginine and histidine), acidic amino acids (such as aspartic acid and glutamic acid), uncharged polar amino acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar amino acids (such as alanine, valine, leucine, isoleucine, PR is Lin, phenylalanine, methionine and tryptophan), β-branched amino acids such as threonine, valine and isoleucine) and aromatic amino acids (such as tyrosine, phenylalanine, tryptophan and histidine).

Moreover, non-conservative substitution may increase the activity of the protein (e.g., constitutively activated proteins).

In addition, proteins that are functionally equivalent to the protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, you can get ways that use hybridization. In other words, the DNA encoding the protein HMGB1, HMGB2, or HMGB3 of the present invention, as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or 16, or a fragment used as a probe and then allocate DNA, which can gibridizatsiya with them. The hybridization reaction, carried out under strict conditions, leads to the selection of highly homologous DNA as the nucleotide sequence. This increases the likelihood that the selected proteins contain proteins that are functionally equivalent to the protein HMGB1, HMGB2, or HMGB3. Examples of highly homologous nucleotide sequences include sequences having 70% or more, preferably 90% or more identity.

In the specific example, the term "stringent conditions" refers to conditions of hybridization of 6×SSC, 40% formamide at 25°C and subsequent washing in 1×SSC at 55°C. Page the visitor is dependent on such conditions, as the salt concentration, the concentration of formamide or temperature; however, experts in this field know how to set the conditions to obtain the necessary rigor.

Using hybridization, for example, you can allocate DNA encoding homologues of proteins HMGB1, HMGB2, or HMGB3, non-protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15.

Proteins that are functionally equivalent to the protein containing the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, normally have a high homology with the amino acid sequence SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15. The term "high homology" refers to identity sequence comprising at least 30% or more, preferably 50% or more, more preferably 80% or more (for example, 95% or more). The identity of nucleotide sequences and amino acid sequences can be determined using the website search homology via the Internet (e.g. search for homology, such as FASTA, BLAST, PSI-BLAST, and SSEARCH, can be used in DNA Data Bank of Japan (DDBJ) [examples of which include the search page homology (Search and Analysis on the web-site DNA Data Bank of Japan (DDBJ); http://www.ddbj.nig.ac.jp/E-mail/homology-j.html]). Moreover, the search using BLAST can be performed through the web-site of the National Center for Biotechnology Information (NCBI) (examples of which included the Ute page BLAST on the home page of the web site NCBI; http://www.ncbi.nlm.nih.gov/BLAST/; Altschul, S.F. et al., J. Mol. Biol., 1990, 215(3): 403-10; Altschul, S.F. & Gish, W., Meth. Enzymol., 1996, 266: 460-480; Altschul, S.F. et al., Nucleic Acids Res., 1997, 25: 3389-3402)).

For example, when calculating the identity of amino acid sequences using Advanced BLAST 2.1 value identity (%) can be obtained as follows: as programs are using blastp, the expected value set to 10, all filters set to OFF, as the use matrix BLOSUM62 and the penalty for missing the penalty for skipping residue and the ratio of the lambda set at 11, 1 and of 0.85, respectively (default settings) (Karlin, S. and S.F. Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68; Karlin, S. and S.F. Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7).

Proteins of the present invention or proteins functionally equivalent, can be a protein, subjected to various modifications, such as physiological modification of sugar chains and the like, tagging fluorescent or radioactive substances or merge with other proteins. Particularly in recombinant molecules, described below, the modification of a sugar chain can vary depending on the hosts used for expression. However, even if there are differences in the modification of a sugar chain, all proteins with properties similar to the properties of the protein HMGB1, HMGB2, or HMGB3, described in this document represent proteins HMGB1, HMGB2 HMGB3 of the present invention or proteins, functionally equivalent.

Proteins HMGB1, HMGB2, or HMGB3 can be obtained not only from the materials of living organisms, but also in the form of recombinant proteins by incorporating the genes that encode these proteins, in expressing appropriate system. For protein HMGB1, HMGB2, or HMGB3 methods of genetic engineering, the above DNA that encode proteins HMGB1, HMGB2, or HMGB3, you can embed the appropriate expressing the system, and then they can be expressed. Examples of systems owner/vector applicable to the present invention, include expressing vector pGEX andE. coli. In the case of pGEX foreign genes can be expressed as a fusion with glutathione-S-transferase protein (GST) Gene, 67: 31-40, 1988). pGEX, which is embedded in the gene encoding protein HMGB1, HMGB2, or HMGB3, introducing a strain ofE. colisuch as BL21, by heat shock, incubated for a suitable period of time, and then add isopropylthio-β-D-galactoside (IPTG) for induction fused with GST protein HMGB1, fused with GST protein HMGB2, or fused with GST protein HMGB3. Because GST according to the present invention is adsorbed to glutathione-sepharose 4B, the expression product can easily be distinguished and clear affinity column chromatography.

In addition, as systems owner/vector to obtain a recombinant protein HMGB1, HMGB2, or HMGB3 you can use the following. P is IDE all, when the owners use bacteria expressing vectors for slit proteins using his-tag tag, HA-tag, FLAG-tag, and is similar to them, are commercially available. As for the yeast, it is known that yeast belonging to the genusPichia,are effective for the expression containing the sugar chain proteins. From the point of view of adding sugar chains are also suitable expressing the system using baculovirus vector with insect cells as host cells (Bio/Technology, 6: 47-55, 1988). In addition, when using mammalian cells, transfection of the vector is performed using promoters such as CMV, RSV and SV40. Any of these systems host/vector can be used as a system for expressing proteins HMGB1, HMGB2, or HMGB3. Furthermore, genes can also be entered using viral vectors such as retroviral vectors, adenoviral vectors, and vectors based on adeno-associated viruses.

Thus obtained proteins of the present invention can be distinguished from the internal contents of the cell or outside the cell (environment etc), and they can be cleaned as proteins, which are essentially pure and homogeneous. Proteins can be separated and cleaned using the methods of separation and purification, which are usually used in isout in the purification of proteins, and they are not specifically limited. For example, proteins can be separated and cleaned by appropriate selection and combination of chromatographic columns, filters, ultrafiltration, vysalivaniya, deposition solvent, solvent extraction, distillation, thus, SDS-polyacrylamide gel electrophoresis, electrophoresis with isoelectric focusing, dialysis, recrystallization, etc.

Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography (Marshaket al., Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Cold Spring Harbor Laboratory Press, 1996). These types of chromatography can be performed using liquid chromatography, such as HPLC and FPLC.

Moreover, proteins of the present invention preferably are essentially purified proteins. In this document, the term "essentially purified" means that the purity of the protein of the present invention (the proportion of the protein of the present invention in the total protein components) is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 100% or close to 100%. The upper limit for the expression "close to 100%depends on cleaning methods and analytical methods specialists in this about the region, and his examples are 99,999%, 99,99%, 99,9%, 99% etc.

Moreover, essentially purified protein includes any protein, purified by any cleaning method, provided that the purity of the protein is the same as described above. Examples include, but are not limited to, proteins, essentially purified by appropriately selecting and combining the above chromatographic columns, filters, ultrafiltration, vysalivaniya, deposition solvent, solvent extraction, distillation, thus, SDS-polyacrylamide gel electrophoresis, electrophoresis with isoelectric focusing, dialysis, recrystallization, etc.

Cells where released or are secreted proteins HMGB1, HMGB2, or HMGB3 pharmaceuticals of the present invention mainly include all types originating from tissue cellsin vivo. Examples of cells that can be easily assembled and to cultivate are, but they are not limited to, fibroblasts (such as normal skin fibroblasts and cell lines derived from them). Moreover, cells, secreting proteins HMGB1, HMGB2, or HMGB3 can also be obtained as follows. Get the vector of embedding a DNA that encodes a protein HMGB1, HMGB2, or HMGB3, or DNA encoding HMGB1, HMGB2, or HMGB3 associated with DNA that encodes a signal sequence for secretion (ATG CAG ACA GAC ACA CTC CTG CTA TGG GTA TG CTG CTG TGG GTT CCA GGT TCC ACT GGT GAC; SEQ ID NO: 17), in the well-known expressing vector or vector for gene therapy. The resulting vector is introduced into mammalian cells, such as fibroblasts (such as normal skin fibroblasts and cell lines derived from them), insect cells, and other cells. Examples of the DNA encoding the signal sequence for secretion include, but are not limited to, DNA described above sequences. There are no specific limitations to the type of animal from which these cells occur, although it is preferable to use cells from the type of animal to which an animal target, which is subjected to the regeneration of tissues, cells of the animal target, or cells originating from a close relative of the animal target, which is subjected to the regeneration of tissues.

DNA that encode proteins HMGB1, HMGB2, or HMGB3 pharmaceuticals of the present invention can be a cDNA, genomic DNA, natural DNA or artificially synthesized DNA, provided that they encode protein HMGB1, HMGB2, or HMGB3. DNA that encode proteins HMGB1, HMGB2, or HMGB3, usually contained in the pharmaceuticals of the present invention in the form of built-in vectors (such as vectors in gene therapy).

Examples of vectors for gene therapy of the present invention include, but are not limited to, plasmids the e vectors, retroviral vectors, lentiviral transfer vectors, adenoviral vectors, vectors based on adeno-associated virus vectors based on Sendai virus, vectors based on shell Sendai virus vectors based on the human papilloma virus. The gene therapy vectors can contain a promoter DNA sequence, which effectively induce the expression of genes, factors that regulate the expression of genes and molecules, which are necessary to maintain the stability of DNA.

The pharmaceutical agents of the present invention may also contain partial peptides of the protein HMGB1, HMGB2, or HMGB3, which have activity involvement of bone marrow cells in the peripheral blood from the bone marrow; cells secreting these partial peptides, or vectors, in which are incorporated the DNA encoding these partial peptides.

Introduction methods of pharmaceutical products of the present invention is injecting into a blood vessel or muscle. Specific examples of routes of administration include the introduction by injection. For example, the pharmaceutical agents of the present invention can be introduced in a blood vessel or muscle by intravascular injection (intra-arterial injection, intravenous injection or similar) or intramuscular injection.

The method of introduction can wearable denim way to choose according to the age and symptoms of the patient. When injected protein HMGB1, HMGB2, or HMGB3, the dose of the protein on the application you can choose from a range from 0.0000001 mg to 1000 mg per kg of body weight of the patient. Alternative dose can be, for example, in the range from 0.00001 to 100000 mg mg body weight of the patient. With the introduction of cells, secreting proteins HMGB1, HMGB2, or HMGB3, or vectors for gene therapy, in which is embedded a DNA encoding a protein HMGB1, HMGB2, or HMGB3, you can enter them so that the number of HMGB1, HMGB2, or HMGB3 was within the above range. However, the dosage of the pharmaceutical agents according to the present invention is not limited to this.

The pharmaceutical agents of the present invention can be produced according to conventional methods (for example, Remington''s Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A), and they may contain pharmaceutically acceptable carriers and additives. Their examples include surfactants, excipients, colorants, fragrances, preservatives, stabilizers, buffers, suspendresume substances, providing isotonicity tools, binders, dezintegriruetsja substances, lubricants, means providing fluidity, and flavoring, although they are not limited, and can appropriately use other common carriers. Specific examples include light anhydrous silicic acid, is actos, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hypromellose, polyvinyltrimethylsilane, polyvinylpyrrolidone, gelatin, triglyceride fatty acids of medium chain length, polyoxyethylene-hydrogenated castor oil 60, white sugar, carboxymethylcellulose, corn starch and inorganic salts.

Moreover, applying the above extracts of cells or tissues, heparin-binding fractions, protein HMGB1, HMGB2, or HMGB3, cells, secreting proteins HMGB1, HMGB2, or HMGB3, vectors, integrated DNA encoding proteins HMGB1, HMGB2, or HMGB3, partial peptides of the protein HMGB1, HMGB2, or HMGB3, cells secreting these partial peptides; or vectors, which are integrated DNA encoding these partial peptides can be such as specified in (1)to(3)below:

(1) the ways in which bone marrow cells in the peripheral blood from the bone marrow, which include the stage of introduction into a blood vessel or muscle extracts of cells or tissues, heparin-binding fractions, protein HMGB1, HMGB2 and HMGB3, cells that secrete proteins, vectors, integrated DNA encoding the protein, a partial peptide of protein, cells that secrete a partial peptides, or vectors, in which are incorporated the DNA encoding the partial peptide;

(2) application of the extract is in cells or tissues, the heparin-binding fractions, protein HMGB1, HMGB2 and HMGB3, cells that secrete proteins, vectors, integrated DNA encoding the protein, a partial peptide of protein, cells that secrete a partial peptides, or vectors, in which are incorporated the DNA encoding the partial peptide, to obtain pharmaceuticals entered into a blood vessel or muscle involvement of bone marrow cells in the peripheral blood from the bone marrow; and

(3) extracts of cells or tissues, heparin-binding fractions, proteins, HMGB1, HMGB2 and HMGB3, cells that secrete proteins, vectors, integrated DNA encoding the protein, a partial peptide of protein, cells that secrete incomplete peptides and vectors, in which are incorporated the DNA encoding the partial peptide, which is injected into a blood vessel or muscle, for use in the methods of involvement of bone marrow cells in the peripheral blood from the bone marrow.

Also the present invention relates to a method of assessing whether contained in extracts of cells or tissues of the factors that involve the bone marrow cells in the peripheral blood from the bone marrow, which include the following stages:

(a) obtaining extracts of cells or tissues; and

(b) definition of activity in terms of involvement of bone marrow cells in the peripheral blood from the bone marrow extracts, Paul is obtained in stage (a).

If the activity involvement of bone marrow cells in the peripheral blood from the bone marrow at the stage (b) exceeds the activity of the control method can also be used to determine that the extracts of cells or tissues contain factors that involve the bone marrow cells in the peripheral blood from the bone marrow.

In the above method, first of all, cells or tissues dipped in the solvent. These cells are not specifically limited, and examples include originating from tissue cells and cell lines derived from originating from tissue cells (such as HeLa and HEK293, but not limited to, the selected cell, the unselected cells (such as cells existing in the selected tissues), cells in which the introduced DNA encoding a protein HMGB1, HMGB2, or HMGB3. As a fabric, as described above, you can use any fabric, and its examples include, but are not limited to, tissue, skin, fabrics for interior biopsy (surgery) (such as the brain, lung, heart, liver, stomach, small intestine, large intestine, pancreas, kidney, bladder, spleen, uterus, testes and blood and damaged tissue. Moreover, examples of the solvent include, but are not limited to, saline, PBS and TBS. In addition, the time of immersion of the cells or tissue in a solvent site which preferably represents the time, necessary and sufficient for the induction of cell necrosis (usually 24 hours or more), but it is not limited to this. Moreover, in the present invention, the cells or tissue dipped in the solvent, and then the cells or tissue can be extracted from the solvent containing cells or tissue. The method of extraction of cells or tissue of the solvent is not specifically limited provided that the method is well-known specialists in this field.

Next, measure the active involvement of bone marrow cells in the peripheral blood of bone marrow derived extracts of cells or tissues. Control includes, for example, solvent immersion of cells or tissues. The active involvement of the bone marrow cells can be measured, for example, by methods described in the Examples; however, the methods are not limited to.

The active involvement of bone marrow cells in the peripheral blood from the bone marrow can be estimated as follows. The obtained extract of cells or tissues injected intravenously, subcutaneously, intramuscularly or intraperitoneally; through the period of time from one minute to four weeks after administration, preferably over a period of time from one hour to 24 hours after administration, more preferably within 12 hours after the injection, peripheral blood is collected and the population of mononuclear cells in the peri is aricescu assess blood flow cytometry for the measurement of double positive for PDGFRα and CD44 cells, or double positive for PDGFRβ and CD44 cells. The method of analysis activity is not limited to this example.

The present invention also relates to methods of screening extracts of cells or tissues, including the factors that involve the bone marrow cells in the peripheral blood from the bone marrow, which include stages:

(a) analysis of a number of extracts described above assessment of whether extracts of factors that involve the bone marrow cells in the peripheral blood from the bone marrow; and

(b) selection of extracts, which are determined at the stage of (a) as containing factors that involve the bone marrow cells in the peripheral blood from the bone marrow.

The present invention also relates to methods of identifying factors that involve the bone marrow cells in the peripheral blood from the bone marrow, which include purification stage factors using the active involvement of bone marrow cells as an indicator of the extracts, which are identified as containing the factors, methods of assessment and screening described above. Factors that involve the bone marrow cells in the peripheral blood from the bone marrow, can be cleaned using conventional methods of isolation/purification of the protein. There is no limit type allocation method/ocistenie, for isolation and purification of proteins can appropriately select and combine chromatographic columns, filters, ultrafiltration, salting out, precipitation with a solvent, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, electrophoresis with isoelectric focusing, dialysis, recrystallization, etc. Peeled factors can be identified by methods known to experts in this field, for example, mass spectrometry. The identified factors can be used to engage the bone marrow cells in the peripheral blood from the bone marrow. Such factors can be called "candidates for involvement of bone marrow cells in the peripheral blood from the bone marrow or candidates for participation in the involvement of bone marrow cells in the peripheral blood from the bone marrow".

All documents of the prior art cited herein, are incorporated herein as references.

Examples

Later in this document present invention is specifically described using examples, but it should not be interpreted as limited by them.

Example 1

Objective: the Involvement of stem cells in the bone marrow into the peripheral blood using induction factors originating from the bone marrow is stvolovyh cells in the extract of the skin tissue.

Methods: to achieve the above objectives, we conducted the survey in the manner described below.

(1) Receiving inductor originating from the bone marrow stem cells. Loose fragments of skin obtained from 25 newborn mice (aged two days) was immersed in 25 ml of phosphate-saline buffer (PBS), pH of 7.4. After incubation for 24 hours at 4°C, the sample was centrifuged at 440 G at 4°C for ten minutes to remove tissue. The supernatant was collected as the extract of the skin (SE).

Meanwhile, from the skin of newborn mice C57/Bl6 RNA was extracted using Trizol (Invitrogen), and then synthesized cDNA using a set of SuperScript III cDNA Synthesis Kit (Invitrogen). Using this cDNA as template was performed polymerase chain reaction (PCR) to amplify cDNA HMGB1. HMGB1 cDNA was incorporated into plasmid vector for protein expression in mammalian cells, pCAGGS, for expression of the protein in which the N-end amino acid sequence related to the sequence of the Flag-tag (Asp-Tyr-Lys-Asp-Asp-Asp-Lys, SEQ ID NO: 18) (figure 1). The plasmid vector was transfusional in HEK293 (cultivated cell line derived from the kidney cells of a human embryo). Cells were cultured for 48 hours for expression of protein. Each sample of cells expressing the protein HMGB1, and the culture supernatant were incubated at 4°C in ECENA 16 hours, and then centrifuged at 4400×g for five minutes. The supernatant was collected and it was added to the gel with antibody against Flag (Sigma) in an amount of 100 μl to 50 ml of supernatant. The mixture is incubated at 4°C for 16 hours. The gel was collected by centrifugation, and then washed five times by PBS. Then the gel was suirable 3× Flag peptide (final concentration 100 μg/ml). The concentration lirovannomu protein was determined using a set of HMGB1 ELISA Kit (Shino-Test Co.). After lyophilization, the protein concentration was brought to 200 mcg/ml using PBS.

(2) Mice at the age of eight weeks (C57/Bl6) were injected with 500 μl of the above extract skin (SE), or 500 μl of PBS as a negative control group, through the tail vein using a syringe to which is attached a needle for injection 30G 1/2 (figure 2). Six, 12, 24 and 48 hours after administration of the hearts of the mice was collected and 1 ml of peripheral blood under inhalation anesthesia using izoflurana using coated with heparin 1-ml syringe. Each sample was mixed with 3 ml of PBS, and then was carefully layered on 3 ml Ficoll (GE healthcare). The samples were centrifuged using a centrifuge at 400×g at 25°C for 40 minutes. Cells muddy middle layer was collected as the fraction of mononuclear cells. The collected cells were added to 1 ml HLB (Measurement-Biological Laboratories Co., Ltd.), hemolytic cf is DSTV. Cells were incubated at room temperature for five minutes. This hemolytic treatment was repeated twice. After adding 10 ml PBS, the cells were centrifuged at 440×g at 25°C for five minutes. Supernatant was removed and the cells were collected. 1000000 cells were incubated at room temperature for 20 minutes with antibodies, each of which was diluted 100-fold PBS, including PE labeled antibody against mouse PDGFRα (e-Bioscience), PE labeled antibody against mouse PDGFRβ (e-Bioscience), and labeled PerCy5 antibody against mouse CD44 (BD biosciences). After incubation, the cells were centrifuged at 440×g at 25°C for five minutes. The supernatant was removed. To the cells was added 400 μl of PBS containing 1% paraformaldehyde, for the preparation of samples for analysis of flow cytometry.

Mice at the age of eight weeks (C57/Bl6) were injected with 250 µl of mouse HMGB1 (1 μg/μl), or 250 μl of PBS as a negative control group, through the tail vein using a syringe to which is attached a needle for injection 30G 1/2 (figure 4). Through 12 hours after dosing were collected 1 ml of peripheral blood from the hearts of mice under inhalation anesthesia using izoflurana using coated with heparin 1-ml syringe. Each sample was mixed with 3 ml of PBS, and then was carefully layered on 3 ml Ficoll (GE healthcare). The samples were centrifuged using a centrifuge pri×g at 25°C for 40 minutes. Cells muddy middle layer was collected as the fraction of mononuclear cells. The collected cells were added to 1 ml HLB (Measurement-Biological Laboratories Co., Ltd.), hemolytic funds. Cells were incubated at room temperature for five minutes. This hemolytic treatment was repeated twice. After adding 10 ml PBS, the cells were centrifuged at 440×g at 25°C for five minutes. Supernatant was removed and the cells were collected. 1000000 cells were incubated at room temperature for 20 minutes with antibodies, each of which was diluted 100-fold PBS, including PE labeled antibody against mouse PDGFRα (e-Bioscience), and labeled PerCy5 antibody against mouse CD44 (BD biosciences). After incubation, the cells were centrifuged at 440×g at 25°C for five minutes. The supernatant was removed. To the cells was added 400 μl of PBS containing 1% paraformaldehyde, for the preparation of samples for analysis of flow cytometry.

Results: it was shown that double positive for PDGFRα and CD44 cells largely involved in peripheral blood after 12 hours after injection of the extract of the skin (SE) (figure 3). Moreover, it was shown that double positive for PDGFRα and CD44 cells largely involved in peripheral blood 12 hours after the injection of HMGB1 (figure 5).

Example 2

Task: testing, involved whether mesenchymal who twolove cells in peripheral blood by intravenous administration of recombinant HMGB1.

Methods: C57BL6 mice (age eight to ten weeks males) were injected with 400 μl of saline containing 100 μg/ml of recombinant HMGB1 (40 µg HMGB1) or 400 μl of saline separately via the tail vein. After 12 hours the mice were taking peripheral blood. The blood samples were diluted with PBS to a total volume of 4 ml of the Diluted blood samples were layered on 3 ml Ficoll-Paque Plus (GE), placed in centrifuge tubes. Samples were centrifuged at 400 G at 18°C for 40 minutes. The middle layer containing mononuclear cells was transferred into a fresh centrifuge tube and to it was added 45 ml of PBS. The tube was centrifuged at 800 G at 18°C for five minutes. The supernatant was removed. Again, was added 45 ml of PBS and the tube was centrifuged at 800 G at 18°C for five minutes. The supernatant was removed. Derived mononuclear cells were incubated with labeled phycoerythrin (PE) antibody against mouse PDGFRα and labeled fluoresceinisothiocyanate (FITC) antibody against CD44 mouse. Then estimated the increase in the content of double positive for PDGFRα and CD44 cells in the fraction of mononuclear cells using flow cytometry (Facscan; Becton, Dickinson and Company).

Results: it was shown that the number of double positive for PDGFRα and CD44 cells, PDGFRα-positive and CD44-negative cells in the fraction of mononuclear the x cells in peripheral blood was significantly increased after 12 hours after administration of HMGB1 (Fig). Specifically, it was demonstrated that HMGB1 has the active involvement of PDGFRα-positive cells in peripheral blood from the bone marrow. PDGFRα is known as a marker of mesenchymal stem cells.

Discussion: PDGFRα and CD44 are known as surface markers of mesenchymal stem cells from bone marrow, which are for illustrative originating from bone marrow pluripotent stem cells. Mesenchymal stem cells from bone marrow are pluripotent stem cells that can differentiate into nerve cells, epithelial cells or similar, and osteocytes, chondrocytes and adipocytes. Meanwhile, fragments of leather used in this experiment are through the ischemic condition. Thus, there is a gradual necrosis of tissues and intracellular proteins, such as nuclear proteins, and cell surface proteins, are released to the outside. HMGB1 is a protein contained in the extract of the skin. When the facial skin or similar, these proteins serve as a signal to attract originating from the bone marrow stem cells in the transplanted skin. Thus, it is assumed that the functional regeneration of the skin graft skin is achieved by the restoration of the epidermis, Geode what we follicular tissue or similar, due to bone marrow cells. From this experiment, in the present invention has been opened successfully, that originating from the bone marrow stem cells are involved in the peripheral blood intravenous HMGB1 or extract of the skin, as described above. This discovery provides new therapeutic methods for the treatment of intractable diseases with tissue damage, such as heart attack, brain damage, heart attack, broken bones and skin ulcers, which are based on the involvement originating from bone marrow pluripotent stem cells in the peripheral blood. In addition, the cells involved in the peripheral blood can be collected common method of blood collection. Thus, the present invention provides a simpler and more secure ways of collecting originating from the bone marrow stem cells in comparison with the conventional method of treatment of a heart attack brain, in which cells are collected from bone marrow.

Reference example 1

Task: identification of a family of HMGB1 in the extract of the skin and the research activity induction of mesenchymal stem cells.

Methods: the Presence or absence of neonatal extract of the skin of the mouse family HMGB proteins was confirmed using the procedure of Western blotting. Loose fragments of skin from 400 newborn mice was immersed in 400 ml of physiological phosphate-saline buffer (PBS; pH 7,4) and the solution was incubated at 4°C for 24 hours. To remove tissue samples were centrifuged at 440 G at 4°C for 10 minutes and supernatant was collected as the extract of the skin. Ten μl of the obtained extract skin was used as a sample and subjected to SDS-PAGE electrophoresis. Proteins separated in the gel were transferred to PVDF-membrane using the device for blotting (ATTO). The membrane was incubated with PBS containing 3% skim milk and 0.1% Tween20 (S-T-PBS) at room temperature for one hour, and then she was allowed to react with each of the rabbit antibodies against HMGB1 mouse, rabbit antibodies against HMGB2 mouse or rabbit antibodies against HMGB3 mouse, which was diluted 1000 times by S-T-PBS, at 4°C for 16 hours. After the reaction, the PVDF membrane was washed by S-T-PBS five times for 5 minutes. Then, the PVDF membrane was incubated with diluted 2000 times (diluted by S-T-PBS) peroxidase labeled antibody goat against rabbit IgG (GE Healthcare) at 25°C for 1 hour. In addition, after washing by S-T-PBS five times for 5 minutes, the PVDF membrane was allowed to react with the system for detection, Western blotting ECL (GE Healthcare). Film ECL exhibited and demonstrated detection etc is the lack of protein HMGB1, HMGB2 and HMGB3.

RNA was extracted from the skin of newborn mice using Trizol (Invitrogen) and then synthesized cDNA using a set of SuperScript III cDNA Synthesis Kit (Invitrogen). Using this cDNA as the template amplified cDNA HMGB1, HMGB2 and HMGB3 way polymerase chain reaction (PCR). cDNA was incorporated into the plasmid vector pCAGGS for protein expression in mammalian cells, so that they can be expressed proteins with an additional sequence of the Flag-tag (Asp-Tyr-Lys-Asp-Asp-Asp-Lys; SEQ ID: 18) on the N-end amino acid sequence. These plasmid vectors were transfusional in HEK293 (culture cell line derived from the kidney of a human embryo) and were cultured for 48 hours for protein expression. Cells expressing each protein HMGB1, HMGB2 and HMGB3 and culture supernatant, incubated at 4°C for 16 hours and then centrifuged at 4400 g for 5 minutes to collect the supernatant. 100 μl of gel antibodies against Flag (Sigma) was mixed with 50 ml of this supernatant, and then incubated at 4°C for 16 hours. Then centrifugation was performed to collect the gel and washing by PBS five times. In addition, protein was suirable using peptide 3× Flag (final concentration 100 μg/ml). The expression of recombinant proteins investigated by the method of Western blotting using razbavleniem (1000 times diluted S-T-PBS) mouse antibodies against Flag and diluted 2000 times (diluted S-T-PBS) peroxidase labeled antibodies against mouse IgG (GE Healthcare). Activity migration of mesenchymal stem cells in the bone marrow of the mouse in these purified recombinant proteins was evaluated by using the camera of Boyden. Moreover, for studies of drug efficacy means based on the HMGB familyin vivofrom the back skin of C57BL/6 mice aged 8 weeks cut out a circle having a diameter of 8 μm, for the implementation of models of skin ulcers. Each of the purified HMGB1, HMGB2 and HMGB3 (100 ng/ml) was mixed with the same quantity of a solution of hyaluronic acid in a concentration of 1 g/100 ml of PBS, and 100 µl was injected onto the surface of the ulcer. The surface of the ulcer was covered with a transparent adhesive dressing on the wound/protective material Tegaderm (3M Healthcare), to avoid drying out, and the area of the wound was measured over time to determine therapeutic effect.

In addition, to investigate whether does the extract of the skin of man and purified HMGB1 human activity ensure that migration of mesenchymal stem cells from human bone marrow, used the camera Boyden. Human skin, with an area of 1 cm², was immersed in 1 ml PBS, and then incubated at 4°C for 16 hours, and then centrifuged at 440 G at 4°C for 10 minutes. The supernatant was separately collected for use as extract of human skin. Moreover, mesenchymal stem cells human bone marrow (Cambrex) use the Ali as cells, placed in the upper compartment of the chamber Boyden (the analysis of surface antigens by flow cytometry confirmed that these cells are CD105-positive, CD166-positive, CD29-positive, CD44-positive, CD34-negative and CD45-negative. Also with the help of test induction of differentiation revealed that they differentiate into adipocytes, chondrocytes and osteocytes). Moreover, in the lower compartment were placed 100 ng/well of human HMGB1 (R&D), and extract of human skin, diluted 10 times in PBS. As control was used PBS.

Results: Western blotting, revealed bands HMGB2 and HMGB3, as well as band HMGB1. Thus, it was confirmed that the extract of the skin of the newborn mouse contains, in addition to HMGB1 proteins family HMGB2 and HMGB3 (6). Received expressing vectors HMGB1/HMGB2/HMGB3 with Flag-tag added to the N-end of each protein (figure 1). These expressing vectors were transfusional in HEK293 cells, and the expressed proteins were purified using Flag-tag, and to identify these proteins was performed by Western-blotting (Fig.7). Activity migration of mesenchymal stem cells from bone marrow of a mouse was determined using these purified proteins, and the activity was confirmed for all of these proteins (Fig). Size of the ulcers formed on the back of the mouse was measured is the each 7 days, and in groups introduction HMGB1, 2 and 3 was confirmed by a significant effect in the reduction of the area of ulcers compared with a group without injection (Fig.9). As in the case of mouse revealed that HMGB1 person and an extract of human skin have activity induction of migration of mesenchymal stem cells from bone marrow (figure 10).

Discussion: HMGB2 and HMGB3 are known as proteins having high homology with HMGB1. It is also assumed that these proteins have properties similar to HMGB1. It was confirmed that HMGB2 and HMGB3 HMGB family are also extracted from the extract of the free edge of the skin. In addition, he produced recombinant proteins HMGB1/HMGB2/HMGB3, and were also confirmed by their activity against the migration of mesenchymal stem cells from bone marrowin vitroand therapeutic effect on skin ulcersin vivo. It was found that the HMGB family (HMGB1/HMGB2/HMGB3) and recombinant HMGB family in a free cut the skin of newborn mice have activity induction of mesenchymal stem cells from bone marrow and activity of local induction originating from bone marrow stem cells that can differentiate into epithelium, and thus induced group originating from bone marrow cells differentiated into various cells such as epidermal keratinocytes, hair follicles and fibral the texts in the damaged tissue, provide restoration of damaged tissue. Moreover, because of mesenchymal stem cells from bone marrow are multipotent stem cells, the authors of the present invention believe that similarly can be expected therapeutic effects by systemic injection or local administration HMGB family to treat lesions in other tissues, such as tissue damage, such as brain damage, heart attacks and bone fractures.

Moreover, it is known that between human and mouse homology of amino acid sequences for HMGB1 is 98% (213/215), 96% (202/210) for HMGB2, and 97% (195/200) for HMGB3. Thus, it is believed that HMGB person and HMGB mouse have similar activity, and the results showed that the extract of human skin and human HMGB1 have the same activity induction of mesenchymal stem cells from bone marrow, as the extract on mouse skin and mouse HMGB1.

Reference example 2

Task: setting the mode of production of tissue extract containing the factors inducing mesenchymal stem cells in the bone marrow.

Methods: brain, heart, intestine, kidney and liver of the mouse C57BL6 at the age of 6 weeks and the skin of newborn mice was immersed in 1 ml of physiological phosphate-saline buffer (PBS) at a pH of 7.4. The solutions were incubated at °C for 24 hours, and then was centrifuged at 440 G at 4°C for 10 minutes to remove tissue. Supernatant collected by obtaining extracts of tissues. To confirm, does the thus obtained extract activity induction originating from bone marrow mesenchymal stem cells, investigated the activity of migration originating from bone marrow mesenchymal stem cells using camera Boyden. Moreover, we measured the concentration of HMGB1 contained in these samples, using a set of HMGB1 ELISA Kit (Shino-Test). In addition, the extracts of brain tissue, heart and skin were allowed to contact affinity column with heparin and activity induction originating from bone marrow mesenchymal stem cells in associated with the protein fraction was confirmed using camera Boyden.

Results: the extract of mouse brain contained a number of HMGB1, equivalent to the extract of the skin of a newborn mouse. Furthermore, the activity of the induction of mesenchymal stem cells from bone marrow were also observed in the brain of the mouse, as in the skin. Although the extract of mouse intestine and extract the hearts of mice contained little HMGB1, also observed activity induction of mesenchymal stem cells from bone marrow. Moreover, associated with a column of heparin fractions of brain and mouse heart mouse, and that is also associated with a column of heparin fraction of mouse skin, showed activity induction of mesenchymal stem cells from bone marrow (11). Table 1 presents the results of measuring the concentration of HMGB1 activity and induction of mesenchymal stem cells from bone marrow in each of the extracts of tissues of the mouse.

Discussion: the developed method, which HMGB1 can convenient the m image can be extracted not only from the skin, but also from the brain by direct immersion of these organs in physiological buffer. This method is also applicable to other organs such as the liver and kidney. Moreover, although extracts from the bowels and heart contain little HMGB1 was observed activity induction of mesenchymal stem cells from bone marrow. This indicates that these extracts contain another substance(a), inducing mesenchymal stem cells from bone marrow, in addition to HMGB1. Such substances contained in these extracts, the source is present in each tissue, and believe that they are under physiological conditions attract mesenchymal stem cells from bone marrow to the injured tissue when the tissue is damaged. Using the present invention, a new way comfortable and functional extraction from various organs of many compounds inducing mesenchymal stem cells in the bone marrow, including HMGB1. In addition, also developed a method of purification of substances inducing mesenchymal stem cells from bone marrow, from the extract of the tissue using a link to a column of heparin. These compounds having activity induction of mesenchymal stem cells from bone marrow, can be cleared from the brain and heart, as well as of skin, using a column of heparin.

A DSS is adjustment example 3

Task: establish extraction method : activators migration of mesenchymal stem cells from cultured cells.

Methods: originating from the kidney of a human embryo cell line HEK293 and the cell line carcinoma human cervical HeLa, each, were cultured in D-MEM containing 10% fetal calf serum (Nacalai). These cells were washed by PBS, and then 10⁷cells were immersed in 5 ml of PBS (Nacalai) at 4°C for 16 hours. The solution was centrifuged at 440 G (gravitational acceleration) at 4°C for 5 minutes and then the supernatant was collected. Mesenchymal stem cells human bone marrow was placed in the upper compartment of the chamber Boyden and diluted 5 times (by DMEM) extract of the cells was placed in the bottom compartment to confirm the migratory activity of mesenchymal stem cells from human bone marrow.

Results: as the extract of HEK293 and HeLa extract showed similar activity against migration of mesenchymal stem cells from bone marrow (Fig).

Discussion: the activators migration of mesenchymal stem cells from bone marrow extracted successfully convenient way immersion cultivated cells in PBS.

Reference example 4

Objective: to study whether to induce regeneration of nerve cells by creating models Def the KTA brain of mice which is injected purified on a column of heparin fraction of the extract of the skin by way of a slow-release locally in the injury area, enabling the migration of stem cells contained in the myeloid system, locally in the area of corruption.

Ways:

(1) Obtaining purified on a column of heparin fractions of the extract of the skin

Cut a slice of the skin of newborn mice were incubated in PBS (mouse/ml) at 4°C for 16 hours and got an extract of the skin. Extract skin was diluted 10 times in 9 volumes of 20 mm phosphate buffer at pH 7.5 at 4°C. for Pre-equilibration of the column in the column HiTrap Hepalin HP (column volume: 5 ml, GE Healthcare) was poured 20 mm phosphate buffer, pH 7.5 (30 ml). Then the diluted solution was allowed to contact with the column. Then the column washed with 20 mm phosphate buffer at pH 7.5 and 100 mm NaCl (30 ml). For elution of adsorbed proteins was poured into the column 20 mm phosphate buffer with pH 7.5 and 1000 mm NaCl, and fractions were suirable in tubes. Each of adsorbed fractions was assessed by determining the activity of migration originating from bone marrow cells of the mouse by using method with camera of Boyden and collected fraction(s)having activity against migration. Solution(s), with activity, was used as purified on a heparin fraction(s) of the extract of the skin in the following reference example.

(2) generation of mice with suppression of bone marrow

Mice were irradiated with a single dose of x-ray radiation, part 10 G, to obtain mice with bone marrow is suppressed.

(3) bone marrow Transplantation mice with GFP mice with suppression of bone marrow

Bone marrow cells were collected from both femurs and bone tibia GFP mice. These cells were injected into mice with suppression of bone marrow through the tail vein 24 hours after irradiation. The introduction was performed under inhalation anesthesia using izoflurana.

(4) obtaining a model of the defect of the brain (defect of brain tissue) in mice

Mice with suppression of the bone marrow, transplanted bone marrow cells of the mouse with GFP, were subjected to inhalation anesthesia using izoflurana and pentobarbital (45 mg/kg), the mice were intraperitoneally injected with. Mice were fixed to a stereotactic device for brain and spent the middle section of the head with a scalpel. Trepanning was performed on 2.5 mm lateral to the right and 12.5 mm in front of bregma using borax (figa). At a depth of 3 mm from this site, placed and fixed needle 20G Surflow. Then applied negative pressure using a syringe for sucking part of the brain tissue (pigv).

(5) the Introduction of purified on a column of heparin is different fractions of the extract of the skin in the region of the defect brain tissue

Five microlitres purified on a column of heparin fractions of the extract of the skin, dissolved in the adhesive composition for tissue-based fibrin (fibrinogen) (Bolheal (Kaketsuken)), were injected with the above area, and then were injected with 5 µl of the adhesive composition for tissue-based fibrin (thrombin) (Bolheal (Kaketsuken)) using a Hamilton syringe and a 26G syringe (figs). The objective of this action was to ensure the effect of slow release for purified on a column of heparin fractions of the extract of the skin.

(6) assessment of the effect of the regeneration of nerve cells in areas of the defect of brain tissue

For evaluation used mice of the control group and group administration. Determined the parameters for the corresponding periods of time (over time), mice were perfusion of 4% paraformaldehyde, and then they were fixed and cut out the brain. In addition, conducted external fixation in 4% paraformaldehyde. Then it dehydrational 15% and 30% sucrose gradient with getting frozen sections.

Nuclei were stained with a solution of DAPI (4',6-diamidino-2-phenylindole, dihydrochloride) and the cut closed with the inhibitor of photobleaching. The accumulation of GFP-positive cells in the damaged area (the area of the defect brain tissue) was assessed using a confocal laser microscope.

Results: the quality of the military shows the accumulation of GFP-positive cells within 2 weeks and within 6 weeks after injection. The accumulation of GFP-positive cells tends to be higher in areas of damage to the group, and not in the control group, as in 2 weeks (control; fig.13D, fraction purified on a column of heparin extract skin; fige)and 6 weeks (control; fig.13F, fraction purified on a column of heparin extract skin; fig.13G) after injection.

Discussion: the introduction of purified on a column of heparin fractions of the extract of the skin led to the accumulation originating from bone marrow cells in the area of the defect brain tissue, which was in the form of nerve cells. It is known that originate from bone marrow mesenchymal stem cells differentiate into nerve cells and the result showed that purified on a column of heparin fraction of the extract of the skin is able to induce regeneration of nerve cells damaged brain areas. Moreover, it is also applicable to the regeneration of neurons in damaged areas in the brain tissue in ischemic brain diseases and injuries of the brain.

Industrial applicability

The present invention relates to new therapeutic methods for treating intractable diseases with tissue damage, such as heart attack brain, myocardial infarction, bone fractures, and skin ulcer. the moreover, cells involved in peripheral blood, you can get the conventional method of blood collection. Thus, the present invention provides a simpler and more secure ways of taking originating from the bone marrow stem cells in comparison with the conventional method used for treatment of heart attack brain where the cells taken from the bone marrow.

1. Means for involvement of mesenchymal stem cells from bone marrow into the peripheral blood from the bone marrow, which is injected into a blood vessel or muscle and that includes any of the following components:
(a) protein HMGB1;
(b) a cell that secretes a protein HMGB1;
(c) vector, which is embedded in the DNA encoding a protein HMGB1;
(d) protein HMGB2;
(e) a cell that secretes a protein HMGB2;
(f) vector, which is embedded in the DNA encoding a protein HMGB2;
(g) HMGB3 protein;
(h) a cell that secretes HMGB3 protein; and
(i) vector, which is embedded in the DNA encoding a protein HMGB3.

2. The method involving mesenchymal stem cells from bone marrow into the peripheral blood from the bone marrow, including the stage of introduction into a blood vessel or muscle of any of the following substances:
(a) protein HMGB1;
(b) a cell that secretes a protein HMGB1;
(c) vector, which is embedded in the DNA encoding a protein HMGB1;
(d) protein HMGB2;
(e) a cell that secretes white is HMGB2;
(f) vector, which is embedded in the DNA encoding a protein HMGB2;
(g) HMGB3 protein;
(h) a cell that secretes HMGB3 protein; and
(i) vector, which is embedded in the DNA encoding a protein HMGB3.

3. Use any of the following substances (a)-(i) for the manufacture of means for involvement of mesenchymal stem cells from bone marrow into the peripheral blood from the bone marrow, which is injected into a blood vessel or muscle:
(a) protein HMGB1;
(b) a cell that secretes a protein HMGB1;
(c) vector, which is embedded in the DNA encoding a protein HMGB1;
(d) protein HMGB2;
(e) a cell that secretes a protein HMGB2;
(f) vector, which is embedded in the DNA encoding a protein HMGB2;
(g) HMGB3 protein;
(h) a cell that secretes HMGB3 protein; and
(i) vector, which is embedded in the DNA encoding a protein HMGB3.