Method of cell recovery from breast secretion

FIELD: medicine.

SUBSTANCE: there is offered a method of precursor recovery from a human body, including all cells having stem cell characteristics or similar, in particular closely active or multiple active precursors, and such cells are recovered either directly, or indirectly from human breast secretion sampled from the specified human body. This secret can be colostrum, ripened milk or secretions in males and females in a lactation interval, during at least one of the following periods: absence of pregnancy, pregnancy period, lactation period, involution period. Further, the present invention refers to preferable applications of such recovered cells.

EFFECT: improved clinical effectiveness.

24 cl, 8 dwg

 

The technical FIELD

The present invention relates to a method for allocating cells of the human body, as well as applications of these cells.

PRIOR art

Stem cells are defined as the count of clonogenic (giving rise to clones), self-updating precursor cells, which can, through a process of differentiation to give rise to a variety of more specialized types of cells. According to the classical approach it is considered that there are two different types of stem cells. Embryonic stem (ES) cells come from the inner mass of the blastocyst are pluripotent and therefore can give rise to all types of differentiated cells in the body. Other subpopulations of stem cells originate from ES cells and are organ - or tissue-specific. It was thought that these poly potent cells, also known as adult stem cells can only differentiate into tissues of the body from which they are derived. An example of such a poly potent cells are hematopoietic stem cells, which serve for the continuous regeneration of blood cells and immune system.

Stem cells have been isolated from many tissues, these tissues that have a high rate constant cell renewal, such as blood, umbilical cord blood, bone marrow, skin tissue of the intestine and mammary gland, to tissues with a low update rate, such as the brain, skeletal muscle and teeth. For a long time there was a theory that regardless of the tissue source of adult stem cells can only differentiate into the tissue from which they were obtained. However, recent studies have shown that when exposed to a new environment organspecific stem cells can overcome this inherent limitation to transdifferentiate in other tissues. For example, it has been shown that neural stem cells could transdifferentiate to become blood cells, bone-marrow-derived stem cells could transdifferentiate to become muscle cells, brain, liver and heart, as obtained from skin stem cells could transdifferentiate to become brain cells. So now theory associated with limitations in the development of organspecific stem cells, is incorrect, and it is possible that these stem cells under appropriate external influences can could transdifferentiate to become another cell type.

In human milk, there is a mix of different types of cells. In milk are detected secretory cells of the epithelium (lakecity) due to their delamination from the basal membrane of the mammary gland due to the pressure associated with the permanent filling and operain the of cancer. The number of lactation reaches approximately 10-20% of the total cell population. Most of the remaining cells contained in human milk, are white blood cells (immune cells such as lymphocytes, macrophages, monocytes, natural killer cells, basophils, eosinophils and neutrophils). There is an opinion that the leukocytes contained in the milk as to protect the mammary gland from infection, and to provide immune protection of the child. To date, it is believed that milk contains only these types of cells.

The INVENTION

Thus, the present invention is to propose a new method of extraction of precursor cells from the body. In this sense, the term "precursor cells" includes all cells with characteristics similar to the characteristics of stem cells, including primarily, but not exclusively, pluripotent or poly potent precursor cells, such, for example, stem cells.

The present invention solves this problem by obtaining such cells directly or indirectly from human secretions of the mammary gland, for example from colostrum, Mature milk or secret, allocated to men or women during the interruption of lactation, during at least one of the following periods: the period of absence of beremend the STI, pregnancy, lactation period, the period of involution. In other words, the authors demonstrate in this paper that the precursor cells may unexpectedly be also found in human lactation milk and that these cells can be used to create fabrics for mother and child. It should be noted that to highlight the corresponding precursor cells can be used not only the secrets of the human mammary gland, but the secrets of the mammary gland of mammals. Using antibodies specific to the cells-the precursors, it is possible to prove that the secret of the breast, that is, for example, human milk does contain precursor cells.

In the first preferred embodiment of the present invention precursor cells extracted from secret of the breast so that the non-cellular components of the secretion of the mammary gland is separated from cellular components, and in particular that the thus obtained cell components are removed cells are not pluripotent or poly potent. Cellular components of the secretion of the mammary gland, in addition to pluripotent cells can optionally contain secretory epithelial cells, leukocytes, and in particular, cells of human origin, such the AK bacterial cells. These cells are not pluripotent, mostly removed from the secretion of the mammary gland.

According to another preferred variant implementation of the present invention for the selection of precursor cells use the secret of the breast received within a duration of lactation, and use the secret of the breast at certain stages of milk ejection, such as stage after commencement of feeding; the stage at the end of individual feeding; lactation phase; preferably the early phase of lactation.

Possible to implement particularly useful and convenient for the practical application of the method of selection of such precursor cells from the milky discharge, if you apply a magnetic granules. To this end the magnetic granules preferably connected with specific to cells-the precursors of antibody that provides adherence of the granules to the cell predecessors.

Typically, the first stage of cellular components washed from the secretion of the mammary gland, in the second stage of cellular components stained with antibodies to markers of progenitor cells, and in the third stage, the precursor cells are separated from other cells by a direct or indirect way (i.e. directly or indirectly) with associated antibodies, preferably, but not iscrucial is but using magnetic granules. For this purpose stained with antibodies precursor cells attached to the granules, preferably to a small iron beads, and precursor cells are extracted with the help of the granules, preferably in the case of small iron pellets, using a magnet, and subsequently the granules, and, if necessary, the antibodies are separated from precursor cells. This can be realized, for example, the choice of pellets, which were provided with specific antibodies associated with granules, and these antibodies specifically associated with cell precursors. To obtain pure cell granules are then separated from the precursor cells that may, for example, be accomplished using enzymes that cleave the bond between the granules and antibodies. If the relationship between the granules and antibodies based on DNA, this splitting can be done using dnaase, and if the relationship between the granules and the antibody contains amino acid chain, you can use protease.

It was unexpectedly found that while normal precursor cells must be cultivated, that is, to grow, to very specific feeding layers (feeders), as, for example, on the supply layer of mouse fibroblasts allocated in accordance with this method, cells were preceded by tennice not need such specific supply layer, and, as a rule, can be grown on other power layers, for example, such as are disclosed within the scope of the specific examples.

More specifically, the allocation method includes the following stages: (i) all human secret of the breast is subjected to centrifugation, which usually gives the top layer of fat underneath the layer rich in protein and carbohydrates, and sediment at the bottom of the cells; (ii) the fraction of the fat and discard the supernatant; (iii) add, for example, a buffer such as (but not limited to, saline solution in phosphate buffer (PBS) and/or salt solution in Tris buffer (TBS), or the environment, such as (but not limited to) Wednesday Williams (Williams) or RPMI medium, and cells (containing not only precursor cells) resuspending buffer/medium and centrifuged as before, preferably repeating this process 3 or 4 times, getting essentially pure sediment cells; (iv) separating the precursor cells from sediment cells.

Department of progenitor cells from the sediment cells preferably carried out through the following stages: (v) sediment cells suspended in the medium, preferably in RPMI medium containing, for example, serum from a cow fetus; (vi) the suspension incubated with (magnetic) granules, which are preferably before this were incubated with specific cells-the precursors of the antibodies is AMI (preferably specific to stem cell antibodies, such murine antibodies IgG), and these antibodies attached to magnetic beads small section of the circuit, for example, of DNA or amino acids, despite the fact that incubation of cell suspension with these magnetic grains is preferably carried out for 15 min at 4°C; (vii) as soon as precursor cells are associated with magnetic granules, containing cells/pellet vitro put the magnet, resulting in associated with granule cell precursors are attracted to the magnet, while not unrelated to each other and remain in the supernatant; (viii) the supernatant is removed, leaving only the precursor cells associated with the granules by means of antibodies to cells predecessors.

You must pay attention to the fact that it is possible to apply other types of granules or even other means of separation, to provide selective binding of granule cells predecessors. These granules must be separated from the precursor cells, and for this purpose preferably can be applied to the following stages of processing: (ix) precursor cells associated with the granules by means of antibodies to stem cells, are removed using a suitable means of splitting, preferably, in the case when antibodies are associated with beads short chain DNA, put the m add dnaase; (x) the granules are removed by applying again the magnet, so that the granules are no longer associated with the stem cells that are attracted to it; (xi) remove the supernatant liquid, now containing the separated precursor cells.

An alternative method of separation based on the specific growth of pluripotent cells, includes the following stages:

(i) In the first stage the whole human secret of the breast is subjected to centrifugation, which gives the top layer of fat underneath the layer rich in protein and carbohydrates, and sediment at the bottom of the cell.

(ii) In the optional second stage precipitate washed with culture medium. Preferably washed cells only RPMI medium.

(iii) In the third stage, cells from the cell sediment is sown in the prepared device for culturing cells in growth medium and incubated. Preferably the incubation is carried out for 10-30 days, more preferably within 14-20 days.

(iv) the Cells are harvested, i.e. they are removed from the substrate, preferably using trypsinization. After that, the captured cells are washed, preferably with a solution of the growth environment.

(v) Collected and washed cells were seeded growth on the drug transformed basal membrane, growth is preferably carried out up to the stage merged monolayer. In this last phase preferably COI is lsout drug solubilizing basal membranes, isolated from murine EHS sarcoma. This can be, for example, the drug Matrigel™, supplied by the firm BD Biosciences.

This method allows to obtain a culture of cells with morphology characteristic of progenitor cells. Cells are not similar to lakecity, and because growth environment ensures the growth of progenitor cells/stem cells/lactation, they may not be bacterial cells.

In addition, the present invention relates to cells-the precursors, which preferably are pluripotent or poly potent cells-precursors (stem cells), obtained by applying the method described above.

In addition, the present invention relates to applications of such precursor cells, for example, for applications ex vivo, in vitro and/or in vivo. Without limiting the scope of the present invention such application may extend to the following specific examples, or a combination of: the creation of tissues or cells for the mother and/or child and/or other individuals; subsequent exposure to methods of gene therapy or intrauterine effects on the fetus; the creation of cells, tissues, glands or organs to treat diseases; subsequent cloning or scientific research; one or more of the group the following tasks: clinic, diagnostics, bioingenieria is, lactonitrile, tissue regeneration, breast cancer, reconstructive surgery, breast cancer, cosmetic surgery improves breast cancer, regeneration and/or surgery exocrine gland.

Further embodiments of the present invention indicated in the dependent claims.

BRIEF DESCRIPTION of DRAWINGS

On the accompanying drawings show preferred embodiments of the invention.

Figure 1 shows the General mixed population of cells of human milk after removal of fat and skimmed milk.

Figure 2 shows stem cells (sc) after their separation from the General population of the cells of the milk. These cells are placed smear on a slide microscope glass and stained with hematoxylin and eosin. On figa and 2b shows a single stem cell (sc). On figs shown a stem cell that is associated with granule Dynabeads® (Db). Granules Dynabeads® (Db) have a size of 4.5 μm, which provides a rough estimate of the size of the stem cells in 6-7 microns.

Figure 3 shows a single stem cells after 1 day after transfer into the culture; after purification of stem cells from the total cell population of milk allocated stem cells can be grown under a variety of conditions of cultivation.

Figure 4: after 1-2 weeks of culturing selected from the total population of stem cells WA who have to undergo the process of dividing (mitosis); in this picture, the arrow indicates a cell undergoing mitosis.

Figure 5: after cultivation for several months, these cells do not exhibit differentiation into cells of other types; this figure shows a stem cell after 2 months of cultivation, which was placed smear on a slide microscope glass and stained with hematoxylin and eosin.

6: after culturing for 2 months, the stem cells were not differentiated; this figure shows the stem cells are isolated using antibodies SSEA-4 and Tra 1-60 (b), after 2 months of cultivation;

cells were washed from the culture medium and placed smear on a slide microscope glass, and then were rendered in confocal microscope using antibodies SSEA-4 and Tra 1-60 (b)conjugated to a fluorescently labeled secondary antibody (goat IgG to mouse antibodies conjugated with AlexaFluor-488).

Fig.7 shows the growth rate of selected stem cells in culture; it is seen that cells divide and proliferate within 4 days; it was found that the number of cells, separated from the Dynabeads® and antibodies SSEA-4, grows in 4 days of cultivation from approximately 50 cells per Cup to 150 cells per Cup; was the Central area of the Cup and applied cross-model calculation; in every culture : the field was N=10 and simply counted the number of cells in individual days; presented in this figure the data are the results of the experiment conducted in 3 repetitions.

Fig shows the monolayer pluripotent cells derived from the secret of the mammal with the use of an alternative method according to the present invention.

DESCRIPTION of embodiments of the INVENTION

Historically method checks whether the alleged stem cells are actually stem cells, was the fact that it is necessary to transplant the suspects cells irradiated sublethal (on the verge of survival) mice. If after that the transplanted cells were fixed and restored any organs in these mice, it was believed that these cells are pluripotent stem cells. But later identification of pluripotent (stem) cells has become possible to carry without a long process of transplantation on the basis of the identification of cellular markers of pluripotent stem cells (e.g., using antibodies Tra 1-60 and SSEA-4; Chemicon International, Temecula, California, US).

After washing all cells from human milk by repeating the process of soft centrifugation, extraction of the supernatant and re-resuspendable cells in buffer or culture medium stem cells were separated from the General population is AI cells in milk (figure 1) using beads Dynabeads according to the manufacturer's instructions. As a buffer, you can use saline solutions in Tris buffer (TBS) or saline solutions in phosphate buffer (PBS). In particular, it is possible to use solutions of TBS containing 10 mm Tris, 150 mm NaCl with a pH close to 7.4. In the case of solutions of PBS, they may contain 1.1 mm KH2PO4, 140 mm NaCl, 4.5 mm Na2HPO4×7H2O and 2.7 mm NaCl, pH can be increased to approximately 7,4. As nutrient media, you can use the environment Williams or RPMI (Poswell Park Memorial Institute)supplied by the company Sigma Aldrich, US under the product number R6504, R7755, R4130 or W4125, W4128, W1878. You can also use the environment RPMI company Gibco (CA, US)No. 11875-093.

Specific to stem cell antibodies, for example, Tra 1-60, TGA 1-81 and/or SSEA-4, all of them are monoclonal and all are supplied, for example, the firm CHEMICON International 28820 Single Oak Drive, Temecula, CA 92590, for the isolation of precursor cells were attached to Dynabeads®. You can also use antibodies to hematopoietic cells CD133 (catalog No. MAV) firm R&D Systems, Inc., CA, US).

Immature hematopoietic stem and precursor cells is isolated from peripheral blood using immunomagnetic principles of cell division. The study group was successfully isolated and cultured CD133 positive cells using magnetic particles conjugated with monoclonal antibodies (system CliniMACS System and reagent AS from the company iltenyi Biotec).

Any specific stem cell antibodies (including SSEA-3, SSEA-1 and Tra 1-81, Oct-4, CD133) incubated with Dynabeads®. Dynabeads® are delivered by the company Dynal AS, and represent a small iron pellets, which through a short chain DNA bound antibodies - mouse IgG. For example, available Dynabeads® under the name Dynal CD34. Before incubate Dynabeads® isolated from milk cells for 15 min at 4°C, carry out incubation Dynabeads® for 1 h at room temperature.

Once the stem cells are associated with Dynabeads, which usually takes approximately 30 minutes to 1 hour, to the lateral wall of the tubes containing cells/Dynabeads®, bring the magnet. Dynabeads® is a homogeneous paramagnetic beads on the basis of polystyrene diameter of 4.5 μm. Dynabeads® (together with the attached stem cells) are attracted to the magnet, and unbound cells are not drawn and remain in the supernatant. Then the supernatant is removed, leaving only the cells that are associated with Dynabeads® by antibodies to stem cells. Cells associated with Dynabeads® by antibodies to stem cells, is removed by adding dnaase, destructive short chain DNA. This call frees the buffer, and it is part of a set Dynal kit containing 62500 units/ml dnaase (from 15,000 to 20,000 units in a bottle, as indicated in the instructions). Dynabead® remove, again applying the magnet, which attracted Dynabeads®are not associated with more stem cells. Now the supernatant contains the detached stem cells. These stem cells can now be used for any subsequent use, as described above and below.

Retrieved precursor cells (stem cells) can be grown to supply the cellular layer fibroblasts of mouse embryo in the environment of the Needle in the modification Knockout-Dulbecco. Usually, the cultivation can be carried out at a temperature of 37°C.

Examples of the use of feeder cells:

- Long reproduction of stem cells from human embryos currently carried out by co-culture with primary fibroblasts mouse embryo, which serve as feeding cells.

- Accelerate the formation of cultured epithelium by using as a layer of feeder cells treated with ultrasound expressing cells hedgehog.

- Cells in the bone marrow of the adult support long germination in the culture of stem cells of a human embryo.

For proliferation of embryonic stem cells into endothelial cells and their maturation in models in vitro necessary supply cells OR.

- Selective germination and continuous culture of macrophages from the blood of adult pigs. macrophage from the blood of adult pigs were selectively parsevals and continuously cultivated directly in the environment, covering supply layer of mouse fibroblast STO.

- At the base and characterization of a new line of human immature cells megakaryoblastic leukemia (M-IOC) for the viability of cells required fibroblasts. Was founded as a new dependent fibroblast line human immature cells megakaryoblastic leukemia (M-IOC) from the bone marrow of the girl with the sharp megakaryoblastic leukemia, and it was found that the cell growth is completely dependent on the presence of human fibroblasts HEL-O, obtained from the lungs of the embryo.

For in vitro culture of cells of embryonic blastodisc of the pig blastocyst as a feeding layer using pre-irradiated fibroblasts fetal pig G30.

- It was found that cell acute lymphocytic leukemia (ALL) babies and children survive mainly in co-culture with cell line cloned fat cells of the endothelium (14F1.1) from the bone marrow of the mouse and show extensive growth in the presence of murine stromal cells. These cells are ALL strictly depended on the presence of murine stromal clone 14F1.1 and had lost the ability to proliferation in the absence of adipocytes endothelium or in the presence of feeder cells.

The growth of cells

It was found that the number of cells extracted using Dynabeads® and antibodies SSEA-4, the 4 day increases from approximately 50 cells per Cup to 150 cells per Cup (see Fig.7).

Methods

Preparation of cells

Whole milk (150 ml), centrifuged for 15 min at 2000 rpm, to collect cells. Sediment cells resuspended approximately 4 ml of TBS with 1% BSA and centrifuged again for 10 min Final precipitate resuspended in 200 μl of RPMI medium with 1% serum fetal cow (FCS).

Preparation of granules

Pellets were washed 3 times in RPMI medium with 1% serum fetal cows.

The pellets were covered with primary antibody (1 μl 500 μl of TBS with 1% BSA) and incubated for 1 h at room temperature with careful stirring.

The coated granules were separated, washed 3 times with 1 ml TBS with 1% BSA and transferred into a clean tube. The last washing was made in 1 ml of RPMI medium with 1% FCS.

The selection of cells

To the pellet was added 200 μl of a preparation of cells and incubated for 30 minutes at approximately 4°C. After incubation the unbound fraction is discarded.

For complex cells with beads add 200 ál of RPMI medium with 1% FCS and then subjected to its gentle pipetting to remove residual unbound substances.

To 200 μl of complex cells with granules add 5 ál of releasing buffer and incubated with careful stirring for 15 min at 37°C. the Complex granule cells subjected to intensive pipetting to facilitate release of the cells.

Izvlecheny the cell suspension (200 µl) was transferred into a clean Eppendorf tube. This cell suspension can be directly entered into the system for cultivation.

Method of cultivation

Cells plated on coated collagen Cup for cultivation. After a period of subsidence and linking cells supernatant carefully drained to remove impurities, and add fresh medium. Then the culture is incubated in the dish at 37°C in an incubator with 5% CO2, replacing the medium every 2 days.

Media for culturing

Wednesday Williams E Williams E), supplemented with 10% fetal serum cows; 10-7M dexamethasone (Sigma); 2 mmol/l glutamine; pre-made mixture of ITS+containing insulin (at 6.25 µg/ml), transferrin (at 6.25 µg/ml), selenic acid (at 6.25 ng/ml), bovine serum albumin (1.25 mg/ml) and linoleic acid (5,35 µg/ml) from the company Becton-Dickinson, Bedford, MA. Antibiotics: penicillin/streptomycin 5000 µg/ml Fungizone (250 mg/ml).

Antibodies

Examples of some of the antibodies that are potentially suitable for use in this system are (all are delivered by the company CHEMICON International 28820 Single Oak Drive, Temecula, CA 92590):

SSEA-1 (catalog No. MAV)

SSEA-3 (MAB4303)

SSEA-4 (MAD4304)

Tra 1-60(MAB4360)

Tra 1-81 (MAB4381)

antibodies to hematopoietic cells CD133 (catalog No. MAV) from firm R&D Systems, Inc.

Buffers

TBS: 1% BSA, 10 mm Tris, 150 mm NaCl

RPMI with 1% FCS

PBS: 1.1 mm KH2PO4, 140 mm NaCl, 4.5 mm Na2HPO4×7H2 O

After purification of stem cells from human milk using antibodies to the external markers of stem cells (Tra 1-60 and SSEA-4), associated with Dynabeads®, did swabs stem cells on microscope slides and stained stem cells with hematoxylin and eosin (figure 2). Using the same purified stem cells, the authors were able to cultivate cells (figure 3) and to demonstrate that these cells are able to share (figure 4). After prolonged cultivation, the authors were able to paint these cells with hematoxylin and eosin and to demonstrate that these cells remain stem cells, because they are after a few months of cultivation continue to communicate with antibodies to stem cells (Tra 1-60 and SSEA-4), as revealed by confocal microscopy (6). The growth of individual cells was also confirmed (see Fig.7).

An alternative method of allocation/cultivation of pluripotent cells is the use of specific combinations of growth medium, through which it is possible to grow a monolayer of pluripotent cells, which certainly don't look like lakecity (see Fig). Indeed, grown in culture, the cells are physically identical, for example, cultures of liver stem cells. Additional identification of these cells using cell markers they Express the shape, and on the activity of their genes.

An alternative method of selection or selective growth can be carried out as follows.

Cooking cups coated with Matrigel™

Matrigel (commercial drug converted basal membrane, BD Matrigel™, the company BD Biosciences, catalog No. 354234) was slowly thawed in an ice bath. The pipette tips and tablets for cultivation before using cooled and kept on ice. Selected aliquots of 50 μl of Matrigel, transferred them from the tip of the pipette into each of the cells in the microplate for 24 cells with a flat bottom, and incubated on ice for 30 minutes After that, the tablet additionally incubated for 30 min at 37°C for bonding of the gel. After binding to each cell was added in 1 ml of growth medium and returned the tablet in the unit to use (for a period not exceeding 5 days).

The cultivation and preparation of cells

Approximately 50 ml of their residual milk (hind milk women breast milk with a high content of fat, cream-colored; it gives the child the main part of the calories and contributes the most to gain mass) was centrifuged to transfer cells in the sediment (see details above). The precipitated cells were washed 2 times only RPMI medium (RPMI 1640 Medium, firm Gibco, catalog No. 108-36).

After that, cells were sown on a specially treated to to the steverivonia cells in plastic culture Cup in the growth medium (the composition of the growth medium for cell cultures: RPMI 1640; serum fetal cows 20%; insulin, 5 μg/ml cholera toxin 50 ng/ml; hydrocortisone 0.5 μg/ml; 2-fold concentration of penicillin-streptomycin-Fungizone) and incubated for approximately 14-20 days.

After incubation took the Cup with large colonies and close to the confluence of the monolayer and removed cells trypsinization (Trypsin-EDTA 1x, firm JRH Bioscience, catalog No. 59218).

The collected cells were washed 1 time in growth medium and were sown in coated with Matrigel™ cell tablet in the amount of approximately 1500 cells/ml After approximately 14 days it was possible to observe areas of merged cells.

Description

Morphology typical of epithelial cells in the sense that develop fused layers of cells, and cells in the colonies closely associated. The fact that obtained from breast milk cells cultured in this way, develop in primary culture and form extensive round colonies with well-defined borders, strongly suggests that the cells have characteristics similar to the qualities of progenitor cells. What cells are undifferentiated and many of them reveal a high ratio of the volume of the nucleus and cytoplasm, even more evidence to identify them as such cells predecessors.

Used specific materials

Cholera Tox is n (LIST BIOLOGICAL LABORATORIES, catalog No. 101)

Serum of cow fetus (Fetal Bovine serum, Invitrogen, catalog No. 10099141)

Hydrocortisone (Sigma, catalog No. NO)

Insulin (Sigma, catalog No. 19278)

Penicillin/streptomycin/Fungizone (Scott Scientific, catalog No. E)

The microplate MICROPLATE (IWAKI, catalog No. 3820-024)

Cup for cell culture (Corning, catalog No. 430165)

Due to the adaptability to the external environment of these pluripotent stem cells, these bins can be used for many different applications. For example, these cells can:

- can be used to create fabrics, mainly for mothers and children (and, in principle, for other individuals), including subsequent impacts methods of gene therapy or intrauterine effects on the fetus, and to create cells, tissues, glands or organs to treat diseases. All of the above includes their use in scientific research, clinical, diagnostic or commercial applications. And may also include the creation of biological compounds, including cells, cell fragments, cellular secretions, cellular fractions, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors and cytokines. In addition, the above application of the cell can is to enable the creation of cells as a stage, prior to the creation of tissue, glands or organs, or as a stage subsequent to the creation of tissue, glands or organs to treat diseases, tissue regeneration, improve the body or cosmetic applications for the following tissues: olfactory, auditory, ocular, lymphatic, immune, hematopoietic, endocrine, exocrine, intestinal, gastro-intestinal, the fabric of Meyerovich plaques, islets of Langerhans, skeletal, muscular, connective, vascular tissue, blood, skin tissue, hair, nails, breast cancer, brain and Central nervous system, liver, heart, lung, kidney, bone, pancreas, reproductive organs;

- to store for future use. Subsequent use of these stem cells, or differentiated or dedifferentiated cells includes storing these stem cells for future use, as described below. This includes storage for later use in research, clinical, diagnostic or commercial applications;

- used for cell cultures, for example, for the reproduction of the same stem cells or to differentiate or dedifferentiate these stem cells into other types of cells. This includes their use in scientific research, clinical, diagnostic the x or commercial applications;

- be used for cloning. Subsequent use of these stem cells, or cells differentiated or dedifferentiated of these stem cells may enable the application to create clones or embryos, or whole animals. This includes their use in scientific research, clinical, diagnostic or commercial applications;

to primeneniia for scientific research. Subsequent use of these stem cells, or cells differentiated or dedifferentiated of these stem cells may enable the use in scientific research. This may include the creation of biological compounds, including cells, cell fragments, cellular secretions, cellular fractions, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors and cytokines. In addition, the above application of the cells may include cells as a precursor tissue, glands or organs, or as a stage subsequent to the creation of tissue, glands or organs to treat diseases, tissue regeneration, improve the body or cosmetic applications for the following tissues: olfactory, auditory, ocular, lymphatic, immune, hematopoietic, endocrine, exocrine, intestinal, jeludochno-intestinal, the fabric of Meyerovich plaques, islets of Langerhans, skeletal, muscular, connective, vascular tissue, blood, skin tissue, hair, nails, breast cancer, brain and Central nervous system, liver, heart, lungs, kidneys, bones, pancreas, reproductive organs;

- be used for clinical, diagnostic or commercial applications. Subsequent use of these stem cells, or cells differentiated or dedifferentiated of these stem cells may enable their use for clinical, diagnostic or commercial applications. The above application of the cells may include the creation of biological compounds, including cells, cell fragments, cellular secretions, cellular fractions, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors and cytokines. In addition, the above application of the cells may include cells as a precursor tissue, glands or organs, or as a stage subsequent to the creation of tissue, glands or organs to treat diseases, tissue regeneration, improve the body or cosmetic applications for the following tissues: olfactory, auditory, ocular, lymphatic, immune, hematopoietic, endo is rinna, exocrine, intestinal, gastro-intestinal, the fabric of Meyerovich plaques, islets of Langerhans, skeletal, muscular, connective, vascular tissue, blood, skin tissue, hair, nails, breast cancer, brain and Central nervous system, liver, heart, lungs, kidneys, bones, pancreas, reproductive organs;

- be used for bioengineering. Subsequent use of these stem cells, or cells differentiated or dedifferentiated of these stem cells may enable their use to create the body of any other cell type. These cells, tissues or organs can then be used in the cosmetic/reconstructive surgery, transplantation of organs/tissues or the creation of cells/tissues/organs for a third partner. The above application of the cells may include the creation of biological compounds, including cells, cell fragments, cellular secretions, cellular fractions, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors and cytokines. In addition, the above application of the cells may include cells as a precursor tissue, glands or organs, or as a stage subsequent to the creation of tissue, glands or organs to treat diseases the project, tissue regeneration, improve the body or cosmetic applications for the following tissues: olfactory, auditory, ocular, lymphatic, immune, hematopoietic, endocrine, exocrine, intestinal, gastro-intestinal, the fabric of Meyerovich plaques, islets of Langerhans, skeletal, muscular, connective, vascular tissue, blood, skin tissue, hair, nails, breast cancer, brain and Central nervous system, liver, heart, lungs, kidneys, bones, pancreas, reproductive organs;

- be used to lactonitrile. Subsequent use of these stem cells, or cells differentiated or dedifferentiated of these stem cells may enable their use for producing biological components of milk, including cells, cell fragments, cellular secretions, cellular fractions, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors and cytokines;

- be used for the regeneration of the tissues of the breast. Subsequent use of these stem cells, or cells differentiated or dedifferentiated of these stem cells may enable their use to create the breast tissue;

- be used for reconstructive surgery of the breast (b is a hundred). This regenerated tissue, as described above, can then be used for reconstructive surgery of the breast;

- be used for cosmetic breast surgery. This regenerated tissue, as described above, can then be used for cosmetic surgery of the breast;

- be used for regeneration and/or tissue surgery exocrine glands. Subsequent use of these stem cells, or cells differentiated or dedifferentiated of these stem cells may enable their use for tissue exocrine glands, which in turn can be used to repair or replacement of exocrine glands;

- be used to create biological compounds, including cells, cell fragments, cellular secretions, cellular fractions, nucleotides, deoxyribonucleic acids, amino acids, proteins, glycoproteins, carbohydrates, lipids, hormones, growth factors and cytokines.

1. The allocation method from the body of progenitor cells, including all cells with characteristics similar to the characteristics of stem cells, and these cells are obtained directly or indirectly from human secretions of the mammary gland derived from the specified human body, and the secret can be colostrum, Mature mol is kOhm or selection of men or women during the interruption of lactation, during at least one of the following periods: the period of absence of pregnancy, pregnancy, lactation period, the period of involution.

2. The method according to claim 1, characterized in that the precursor cells are pluripotent or poly potent.

3. The method according to claim 1, characterized in that the precursor cells isolated from the secretion of the mammary gland so that the non-cellular components of the secretion of the mammary gland is separated from cellular components.

4. The method according to claim 3, characterized in that the cellular components of the removed cells are not pluripotent or poly potent.

5. The method according to claim 1, characterized in that the secret of the breast removed secretory epithelial cells, leukocytes, and particularly cells of human origin, such as bacterial cells.

6. The method according to claim 1, characterized in that the selection of precursor cells use the secret of the breast received within a duration of lactation, using the secret breast cancer at certain stages of milk such as stage after commencement of feeding; the stage at the end of individual feeding; lactation phase; preferably the early phase of lactation.

7. The method according to claim 1, characterized in that the selection of precursor cells use magni the data granules.

8. The method according to claim 1, characterized in that the first stage of cellular components washed from the secretion of the mammary gland, in the second stage, these cellular components stained with antibodies to markers of progenitor cells, and in the third stage, the precursor cells are separated from other cells directly or indirectly through related antibodies.

9. The method according to claim 8, characterized in that the colored antibody precursor cells is associated with the granules, preferably with a small metal pellets, and precursor cells secrete through the granules, preferably in the case of small iron pellets by using a magnet, and pellets, as well as, if necessary, and antibodies separated from precursor cells.

10. The method according to claim 9, characterized in that the removal of the granules is carried out by enzymes selected from the following group: DNA-ASE, protease, RNA-ASE.

11. The method according to claim 1, characterized in that the precursor cells are cultivated without the use of a feeding layer of fibroblasts, in particular without the use of a feeding layer of mouse fibroblasts.

12. The method according to claim 1, characterized in that
(i) in the first stage of whole human secret of the breast is subjected to centrifugation, which gives the top layer of fat underneath a layer of rich white the AMI and carbohydrates, and at the bottom of the sediment cells;
(ii) in the second stage, a fraction of the fat and discard the supernatant;
(iii) in the third stage, add a buffer such as (but not limited to, saline solution in phosphate buffer (PBS), and/or saline trio-buffer (TBS), or the environment - such as (but not limited to) the environment of Williams (Williams) or RPMI medium, and the cells resuspended buffer/medium and centrifuged as before, preferably repeating this process 3 or 4 times, getting essentially pure precipitate of cells;
(iv) the fourth phase is separated precursor cells from sediment cells.

13. The method according to claim 1, characterized in that the secret of human breast cancer receive sediment cells and then apply the following stage separation:
(i) sediment cells suspended in the medium, preferably in RPMI medium containing serum bovine fetus;
(vi) the suspension is incubated with magnetic granules, which before this were incubated with specific cells-the precursors of antibody, preferably with specific stem cell antibodies, similar to mouse IgG antibodies, and these antibodies attached to magnetic beads small DNA chain, while incubation of cell suspension with these magnetic grains is preferably carried out for 15 min at 4°C;
(vii) once the cells-precursors is anniki be associated with magnetic granules, it contains cells/pellet vitro put the magnet, resulting in associated with granule cell precursors are attracted to the magnet, whereas the unbound cells are not drawn and remain in the supernatant;
(viii) the supernatant is removed, leaving only the precursor cells associated with the granules by means of antibodies to cells predecessors.

14. The method according to item 13, wherein the following specified stages using the following stages:
(ix) precursor cells associated with the granules by means of antibodies to stem cells, are removed using a suitable means of splitting, preferably, in the case when antibodies are associated with the pellets through a short chain DNA by adding dnaase;
(x) the granules are removed by applying a magnet again, so that the granules are no longer associated with the stem cells that are attracted to it;
(xi) remove the supernatant liquid, now containing the separated precursor cells.

15. The method according to claim 1, characterized in that the cells secrete secret of human breast cancer by centrifugation, and then incubated in the growth medium, which provides the growth of progenitor cells/stem Katok/lactation.

16. The method according to item 15, wherein
(i) in the first stage of whole human secret mo is a full-time cancer centrifuged, which gives the top layer of fat underneath the layer rich in protein and carbohydrates, and sediment at the bottom of the cell.
(ii) in the second stage, the precipitated cells are washed with culture medium, preferably only medium RPMI.
(iii) in the third stage, cells from the cell sediment is sown in prepared for cell culture device in a bactericidal and/or fungicidal growth medium and incubated, preferably for 10-30 days, most preferably within 14-20 days.
(iv) the cells are collected, preferably by using trypsinization, and washed, preferably using the growth environment.
(v) the collected cells were seeded growth on the drug converted the basement membrane, it is preferable to progress to stage merged monolayer.

17. The method according to item 16, characterized in that in stage (v) drug use solubilizing basal membranes isolated from murine EHS sarcoma, such as Matrigel™.

18. Precursor cells, preferably pluripotent or poly potent precursor cells obtained using the method according to any one of claims 1 to 17, characterized in that they
represent a count of clonogenic, self-renewing cells capable of through process of differentiation to give rise to more diverse specialized cell types;
represent a count of clonogenic, samoussas the cells, able, through a process of differentiation to give rise to a variety of more specialized types of cells;
possess the ability to bind to specific cells-the precursors of antibody;
have a morphology characteristic of progenitor cells;
have the ability to grow in the growth medium for growth of the progenitor cells;
have the ability to long play;
have the ability to proliferation in primary culture and the formation of extensive round colonies with well-defined borders;
show nedifferentsirovannost;
have a high ratio of the volume of the nucleus and cytoplasm; and
show adaptation to the external environment.

19. The use of pluripotent or poly potent precursor cells obtained using the method according to any one of claims 1 to 17, for their applications, ex vivo and/or in vivo.

20. The application of claim 19, characterized in that it is a application for creation of tissue or cells for the mother and/or child and/or other individuals.

21. The application of claim 19 or 20, characterized in that it is the application for the subsequent impacts of the methods of gene therapy.

22. The application of claim 19, characterized in that it is a application for creation of cells, tissues, glands or organs for l the treatment of disease.

23. The application of claim 19, characterized in that it is the application for the subsequent cloning of animals and/or animal embryos.

24. The application of claim 19, characterized in that it is an application for one or more tasks from the following groups: clinic, diagnostics, bioengineering, lactonitrile, tissue regeneration, breast cancer, reconstructive surgery, breast cancer, cosmetic surgery improves breast cancer, regeneration and/or surgery exocrine gland.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to methods of obtaining deposited lymphokine-activated killer cells (LAKC) for treatment of oncologic diseases. Claimed is method, according to which mononuclear lymphocytes (MNL) are isolated from peripheral blood of malignant effusion by centrifugation on one-step gradient of ficoll with density 1.06-1.08 g/cm3, LAKC are generated and concentrated by successive precipitation of MNL by centrifugation, re-slurring it in RPMI 1640 or DMEM medium with addition of 2-10% AB human serum, interleikin-2 in concentration 0.5-1.0 mlnIU/ml and incubation in CO2 incubator for 48-72 hours, after that suspension of obtained LAKC is deposited in culture medium in amount 2 mln LAKC per 200 ml of medium on sterilised and washed with culture medium porous titanium carriers with 55-60% porosity.

EFFECT: invention allows to increase efficiency of obtained medication for local and local-regional immunotherapy of oncologic patients.

1 ex, 8 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: diploid cell cultures are reduced and collected by propagation in a monolayer in an Eagle's growth medium. The conditioned growth medium is separated from the formed cell layer by sterile bottling. The cell layer is removed, washed by centrifugation. The prepared cell suspension is reduced to the required and bottled in the sterile environment. The conditioned growth medium bottles and cell suspension bottles are consistently single frozen to temperature -20°C and higher and thawed at temperature +25°C and lower, and bottled in the sterile environment. The conditioned growth medium and cell suspension are frozen in open bottles at temperature minus 50°C and lower, kept in the frozen environment for at least 48 hours and then lyophilised in two stages. The first stage involves desorption at temperature minus 50°C to 0°C for at least 20 hours; and the second stage requires sublimation for at least 20 hours at temperature 0°C to 30°C; the finished lyophilised bottles are closed in the sterile environment. The invention allows preparing the lyophilised preparations effective in treatment of deep vast burning wounds, frostbites, degrees III (a, b) and IV subdermal burns, treatment of oral and nasopharyngeal mucosa, are storage-stable at temperature +4°C for at least 12 months.

EFFECT: higher efficacy of the preparations for treatment of diseases.

1 dwg, 3 tbl, 2 cl

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to optimise expression of the antigen protein of the human epidermal growth factor-2 (HER2/neu). To obtain the HER2/neu protein, a nucleic acid synthetic molecule is used, which is codon-optimised for high level of expression of the said protein in a human cell.

EFFECT: invention increases production of the recombinant HER2/neu protein during expression in human cells.

8 cl, 10 dwg, 14 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, specifically to obtaining cell population and can be used in cell transplantology and tissue engineering in order to obtain cell material for restoring nerve tissue damaged due to injury, stroke or neurodegenerative diseases. The method involves isolating a population of stromal cells from fat tissue, immunosorting based on using antibodies against the brain-derived neurotropic factor (BDNF) receptor (TrkB) to obtain an essentially homogeneous sub-population of TrkB-expressing cells for their culturing in a standard medium for mammal cells and differentiation induction through transfer into a medium which contains a neural differentiation inducing substance - BDNF combined with 5-azacytidine or retinoic acid combined with 5-azacytidine.

EFFECT: invention enables to obtain a population of neural differentiation induced stromal cells of fat tissue.

7 cl, 5 dwg, 4 tbl, 6 ex

FIELD: medicine, veterinary science.

SUBSTANCE: invention refers to veterinary science. A culture medium for pre-implantation embryos contains DMEM medium, inactivated blood serum of 6-7-month nanny-goats with blood content of IGF1 and IGF2 making 54.73 and 231.50 nmol/l respectively, glucose, streptomycin and penicillin, in the following ratio, wt %: inactivated blood serum of 6-7-month nanny-goats - 15.0-20.0; glucose - 0.40-0.45; streptomycin - 0.004-0.005; penicillin - 0.003-0.0035; DMEM medium - the rest.

EFFECT: application of the presented culture medium for pre-implantation of embryos leads to prolonged storage stability of an unfrozen embryo, and also improves engraftment of previously implanted embryos when transplanted from donor goats to recipients.

1 tbl, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to produce mammal nucleolar protein SURF-6. The NIH/3T3-174 cell line is obtained through genetic modification of mouse fibroblast line NIH/3T3. Modification is carried out using two successive transfections when the cells reach 70% confluence. The first transfection is carried out with 5 mcg of the pUHrT62-1 plasmid which codes the cell neomycin resistance gene (G-418) and the tetracycline-dependant transactivator, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. The second transfection is carried out with a mixture of 2.5 mcg of the pBI-SURF6 plasmid which bears the EGFP gene (green fluorescent protein) and the SURF-6 protein, and 2.5 mcg of a linear DNA fragment which codes puromycin resistance gene, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. Expression of EGFP and SURF-6 genes is controlled by a promoter which is activated by addition of an inducing substance - doxycycline antibiotic to the culture medium.

EFFECT: obtaining a NIH/3T3-174 cell line capable of producing the SURF-6 protein 10-20 times more than protein content in the initial unmodified fibroblasts when an inducing substance is added.

5 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to produce mammal nucleolar protein SURF-6. The NIH/3T3-174 cell line is obtained through genetic modification of mouse fibroblast line NIH/3T3. Modification is carried out using two successive transfections when the cells reach 70% confluence. The first transfection is carried out with 5 mcg of the pUHrT62-1 plasmid which codes the cell neomycin resistance gene (G-418) and the tetracycline-dependant transactivator, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. The second transfection is carried out with a mixture of 2.5 mcg of the pBI-SURF6 plasmid which bears the EGFP gene (green fluorescent protein) and the SURF-6 protein, and 2.5 mcg of a linear DNA fragment which codes puromycin resistance gene, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. Expression of EGFP and SURF-6 genes is controlled by a promoter which is activated by addition of an inducing substance - doxycycline antibiotic to the culture medium.

EFFECT: obtaining a NIH/3T3-174 cell line capable of producing the SURF-6 protein 10-20 times more than protein content in the initial unmodified fibroblasts when an inducing substance is added.

5 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to a method of producing three-dimensional matrices for tissue-like structures from animal cells. The method involves covalent bonding of histons with the surface of pre-activated biocompatible polymer microspheres made from crystalline dextran. The microspheres with covalently bonded histons are then deposited by centrifuging. Microspheres containing 160-200 mcg protein per 1.0 g are deposited on a substrate surface in amount of 0.5-1.0 mg per 1.0 cm2 and then dried at room temperature. Further, the substrate is washed with a solution at pH 7.5 to remove material which is not bonded to the substrate. The layer of microspheres obtained on the surface of the substrate on which cells are deposited is used as a base for obtaining tissue-like cell structures.

EFFECT: invention increases reliability of the structure and stability of the protein layer of the three-dimensional matrix, simplifies and reduces the cost of the method of producing three-dimensional matrices for tissue-like structures from animal cells.

6 cl, 11 dwg, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and cell biology. It has been established that sensitivity of mouse embryonic stem cells to clinostatting increases depending on cell development stages: from embryonic stem cell colonies to embryoid bodies. It has been shown that embryoid body clinostatting leads to slow down of the beginning of cardiomyocyte differentiation and significant reduction of the amount of cardiomyocytes cut. The invention can also be used in space medicine.

EFFECT: delay of neuronal differentiation on later differentiation stages.

2 cl, 8 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: there is offered a method for preparing a soft tissue filler composition for injection to relief or treat skin damages caused by mechanical or physiological reasons, including the stages as follows: 1) digestion of autologous dermal tissue recovered from autologous skin of a patient by processing with pancreatine/EDTA solution, and cell isolation; 2) cultivation and proliferation of the recovered dermal cells by serum-free cultivation in vitro in a medium containing a growth factor and activation factor for preparing autologous cellular culture material of dermal nature containing dermal fibroblastic stem cells, dermal fibroblastic "transitional" dividing cells, dermal fibroblasts and collagen; 3) centrifugation of autologous cellular culture material of dermal nature for separation of autologous cellular sediment of dermal nature; and 4) slurrying of autologous cellular culture material of dermal nature in glucose solution for injection or any solution for injection to prepare suspension for injection. There is offered a composition prepared by specified method which contains 1×107 to 8×107 cells/ml of autologous cells of dermal origins and 10 to 100 mg/ml of collagen as an effective ingredient.

EFFECT: invention provides therapeutic effect over a short period of time and maintains it for a long time.

13 cl, 7 ex, 8 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to urology and endocrinology, and can be used in treatment of benign prostatic hyperplasia and erectile dysfunction in men. Claimed is application of musk deer musk tincture as medication for treatment and prevention of benign prostatic hyperplasia and erectile dysfunction in men.

EFFECT: invention ensures extension of possibility of treating benign prostatic hyperplasia and erectile dysfunction in men.

1 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: stimulation of physiological or reparative regeneration of cardiac hystiocytes and their endocellular structures is enabled by applying swine adrenal cortex extract. Stimulation of physiological regeneration of cardiac hystiocytes and their nuclei is enabled by 5-week course of swine adrenal cortex extract introduction, every second day in single doses 0.5 ml. Stimulation of or reparative regeneration of cardiac hystiocyte mitochondria in hypobaric hypoxy medium is enabled by double application of swine adrenal cortex extract day before and 30 minutes prior to hypoxy.

EFFECT: effective physiological or reparative regeneration of cardiac hystiocytes.

3 cl, 2 tbl, 6 dwg

FIELD: medicine.

SUBSTANCE: claimed is application of pig adrenal cortex extract as medication for stimulation of growth of spinal marrow motoneurons in mammalian growing organism. Claimed is method of stimulation of spinal marrow motoneuron growth in mammalian growing organism in experiment, which includes introduction of pig adrenal cortex extract on alternate days, with general 5 week course, pig adrenal cortex extract being introduced in single dose 0.5 ml per 100 g of organism body weight.

EFFECT: extending arsenal of medications for stimulation of spinal marrow motoneuron growth.

2 cl, 1 tbl

FIELD: medicine; experimental neurology.

SUBSTANCE: offered method of brain cortex motoneurons growth stimulation for growing mammals in experiment involves daily introduction of adrenal cortex extract within total course 5 weeks. Herewith pig's adrenal cortex extract is introduced in single dose 0.5 ml per 100 g of body weight. Offered is application of pig's adrenal cortex extract as brain cortex motoneurons growth stimulator for growing organism.

EFFECT: extended range of products for brain cortex motoneurons growth stimulation and possibility to use available product.

2 cl, 1 tbl

FIELD: medicine, therapy, reflexotherapy.

SUBSTANCE: the innovation in question deals with carrying out combined intravenous and external laserotherapy in combination with complex medicinal impact upon the body. Additionally, it is important to fulfill monitor intestinal emptying, daily, about 6-8 procedures/course. External laserotherapy should be carried out onto corporal and auricular points. Medicinal therapy deals with introducing T-activin immunomodulators intramuscularly at the dosage of 100 mcg/d and Halavit at the dosage of 100 mg/d, about 10-20 procedures/course. It is important to apply Aciclovir at the dosage of 1 g/d, therapy course corresponds to 14. The intake of Eiconol in capsules at the dosage of 6-9/d, copper derivatives of chlorophyll at the dosage of 0.1g/d and food additive sodium alginate at the dosage of 4 g/d should be carried out during the whole therapy course. Intravenous and external laserotherapy should be fulfilled at about 8-10 procedures/course. The innovation normalizes cellular and humoral immunity, prolongs the aftereffects of therapy conducted and decreases the number of its complications.

EFFECT: higher efficiency of therapy.

3 cl, 4 dwg, 6 ex, 1 tbl

FIELD: medicinal industry.

SUBSTANCE: invention relates to a method for isolating biologically active substance from mammalian pancreas and preparing a medicinal formulation for parenteral administration that can be used in medicine as agent normalizing functions of pancreas. Agent is made as a medicinal formulation for parenteral administration and represents peptide complex with the content of low-molecular fraction from 70% to 890%, molecular mass of its peptide components in the range 74-222 Da and the concentration of polypeptides 2.5-2.9 mg/ml. Agent is prepared from calf pancreas (age is 12 months, not above) or pigs by tissue extraction with acetic acid in the presence of zinc chloride. Method for preparing agent from calf pancreas (age is 12 months, not above) or pigs involves freezing at temperature -40°C (not less), keeping at temperature -20-22°C for two months (not less) and adding 3% acetic acid solution in the volume ratio = 1:5 at temperature 20 ± 5°C. Extraction is carried out at constant stirring and 1% zinc chloride solution is added to the prepared homogenous suspension in the volume ratio = 50:1 followed by cooling at constant stirring to temperature 7-16°C and the following stirring for 1 h in each 4 h in settling for 48 h. Extract is separated from inert substances by separating and acetone is added to extract in the volume ratio = 1:5 and kept at temperature 3-5°C for 4 h. Formed homogenized deposit is precipitated with acetone repeatedly twice (not less) and deposit containing active substance is washed out on Nutch filter with two-fold volume of acetone cooled to temperature 7-16° up to preparing light-gray deposit. Deposit is rubbed through metallic sieve, dried, dissolved in distilled water at room temperature at constant stirring up to the concentration of polypeptides 2.5-2.9 mg/ml. Solution is centrifuged, filtered and subjected for ultrafiltration treatment in device under anti-pressure 1.0 kgf/cm2 (not above) through materials with retaining capacity 15000 Da. Glycocol is added to ultrafiltrate to its final concentration 10-20 mg/ml at pH = 5.6-6.6, solution is subjected for sterilizing filtration under pressure 2.0 kgf/cm2 (not above), poured into ampoules in volume 2 ml and subjected for autoclaving at temperature 120°C for 8 min and under atmosphere pressure 1.1 kgf/cm2. Invention provides optimal technology in isolating peptide complex from calf pancreas (age is 12 months, not above) or pigs with the content of low-molecular fraction from 70% to 90%, molecular mass of its peptide components in the range 74-228 Da, and preparing aqueous solution of extract with the concentration of polypeptides 2.5-2.9 mg/ml. Invention provides both purifying the prepared product from impurities and to enhance its yield. The isolated substance differs from the known substances early prepared from mammalian raw by molecular mass of its peptide components, absence of toxicity and apyretic properties based on the complete removing impurities.

EFFECT: improved preparing method, valuable properties of agent.

3 cl, 2 tbl, 1 dwg, 4 ex

FIELD: experimental biology and medicine, hepatology, nephrology.

SUBSTANCE: invention proposes using extracts of porcine or human fetal adrenal gland cortex for correction of acute renal insufficiency in toxic hepatorenal syndrome caused by carbon tetrachloride (CCl4). For realization for correction method involves carrying out course of subcutaneous injections of extract of porcine or human fetal adrenal gland cortex by 1 injection per 24 h in the dose 2.0-2.5 ml/kg of body mass by total course 10 every day injections. Using the proposed method provides enhancing effectiveness of regenerative processes in kidneys and reducing symptoms of adverse effects.

EFFECT: valuable medicinal properties of agent.

2 cl, 2 tbl

FIELD: medicine, peptides.

SUBSTANCE: invention relates to a method for preparing a peptide complex from animal raw possessing the tissue-specific activity. Method for preparing an agent for maintaining therapy possessing tissue-specific activity involves milling calf or pig organs of age 12 months, not above, addition of 3% acetic acid solution at temperature 20 ± 5°C and extraction is carried out at constant stirring. Then, in 30 min zinc chloride 1% solution is added, mixture is cooled to temperature 7-16°C at constant stirring followed by stirring for every 1 h in each 4 h and settling for 48 h. Extract is separated from inert substances by separation and acetone is added to extract in the volume ratio = 1:5, mixture is kept at 3-5°C for 4 h and formed precipitate is washed out with out with two-fold volume of acetone cooled to temperature 7-16°C, formed precipitate is rubbed through a metallic sieve and prepared end product is dried at temperature 18 ± 2°C. The end product represents a peptide complex with the content of low-molecular peptide fraction from 70% to 90% and with molecular mass of its peptide components in the range 1000-12000 Da wherein this complex comprises amino acids, mineral substances, trace elements and vitamins in biologically bound form. The complex elicits the expressed tissue-specific activity based on the proposed sequence of technological procedures and conditions for their realization involving temperature, temporal and other indices, and by using substances including the parent raw, a definite extractant and others. Peptide component in the prepared complex has no denaturating properties and retains its regulatory properties that suggests its using as an agent for carrying out the maintaining therapy. The proposed agent can be used in medicinal practice as an agent used in carrying out the maintaining therapy.

EFFECT: improved preparing method, valuable medicinal properties of agent.

9 cl, 42 tbl, 14 ex

FIELD: medicine, homeopathy, pharmaceutical industry, pharmacy.

SUBSTANCE: invention relates, in particular, to suppository possessing immunomodulating effect and comprising coneflower, accessory substances and a base, sea-buckthorn oil, arborvitae (thuja) homeopathic essence, baptisia homeopathic essence, Timalin-1C homeopathic trituratium wherein coneflower is a component of homeopathic essence. Suppository comprises anhydrous lanolin and wax as accessory substances and cacao butter as a base wherein components are taken in the definite ration in grams per one suppository of weight 2 g. Proposed suppository possesses the enhanced immunomodulating effect and allows stimulating processes of regeneration and hemogenesis and improves processes of cellular metabolism.

EFFECT: valuable medicinal properties of suppository.

3 ex

FIELD: chemical and pharmaceutical industry.

SUBSTANCE: invention relates to natural immunocorrective preparation Tactivine in spray form. Claimed agent contains Tactivina, vitamin C and 0.14 M sodium chloride solution as solvent in specific component ratio.

EFFECT: agent of improved effectiveness and storage stability.

2 cl, 5 tbl, 4 dwg, 4 ex

FIELD: veterinary science.

SUBSTANCE: one should apply combined introduction of T-activin at the dosage of 5 mcg/kg once daily along with a single introduction of phenbendasol at the dosage of 5 mg/kg. Moreover, T-activin should be introduced for 3 d, and phenbendasol should be introduced simultaneously with the last injection of T-activin. Conditions for injecting immunomodulating agent and certain sequence of its injection along with antihelminthic preparation provide increased resistance to repeated infectioning in animals.

EFFECT: higher efficiency of combined therapy.

2 ex, 2 tbl

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