Agent for treatment of ischemic lesions of tissues and method of its use

FIELD: biotechnology.

SUBSTANCE: invention relates to an agent for treatment of ischemic lesions of tissues, which is a mixture with the ratio of 1.5-3 of two cultures of mesenchymal stem cells, one of which is modified by the genetic structure based on the viral vector which provides hyperproduction of vascular endothelial growth factor, and the other is modified by the genetic structure based on the viral vector which provides hyperproduction of angiopoietin, and a method of treatment of ischemic lesions of tissues by puncture of ischemic tissue, and can be used in medicine.

EFFECT: invention enables to achieve effective vascularisation and repair of ischemic tissue.

4 cl, 4 dwg, 3 ex

 

The invention relates to the field of medicine and biotechnology, namely the means to stimulate vascularization and recovery processes in ischemic tissues, causing increased growth of new blood vessels for the treatment of diseases which are based on insufficient blood supply to tissues, such as critical limb ischemia (CLI), coronary heart disease (CHD), diabetic foot, ischemic stroke, amyotrophic lateral sclerosis, systemic sclerosis, chronic non-healing ulcers, nonhealing wounds.

Human disease caused by poor blood circulation in tissues and organs due to stenoziruyuschego defeat of vessels, such as coronary heart disease, ischemic stroke and ischemic disease of the lower extremities, including due macroangioapthy diabetes are some of the most common causes of morbidity and mortality in developed countries working age despite the introduction in public health practice effective methods of surgical and endovascular revascularization.

In Russia IBS affects about 10 million people of working age and more than one-third of them have stable angina. In 2000, the incidence of CHD in Russia amounted to 425,5 cases per 100 thousand n the village (V.I. Makolkin, FN. Zyabrev "Why it is necessary to reduce the heart rate in the treatment of stable angina": Cardiology, 2006, vol.46, p.88-91); the share of CHD in the Russian Federation accounting for 48% of all cardiovascular diseases. According to foreign authors, the proportion of patients with refractory angina accounts for about 15% of all patients with coronary artery disease.

Stenosing atherosclerosis of the lower extremities, leading to severe ischemia of the extremities and, in extreme cases, to the development of gangrene and amputation is associated with a 3-6-fold increase in cardiovascular morbidity and mortality. The most severe purchase all of the above disease, when combined with diabetes, which is a huge socio-economic burden on society: costs up to 30% of the health budget of Russia (90% of them - on complications of diabetes). According to the materials of European reconciliation Commission (European working group on chronic critical leg ischemia Second European Consensus Document on chronic critical leg ischemia." Eur. J. Vasc. Surg. 1992 V.6 (Suppl. A), P.1-32), out of a million patients with diabetes mellitus KINK diagnosed each year 500-1000 patients. Among the late complications of diabetes in first place on the frequency occupied by vascular macroangiopathies, which is the most frequent cause of morbidity and mortality. The result of vascular macro is myopathie - the syndrome of "diabetic foot", which is manifested in the development of venous ulcers and gangrene of the limbs, causing enormous economic damage to society. The frequency of nontraumatic amputations in patients with diabetes in Russia 10-15 times higher than in the General population, hospital mortality high amputations up to 45-50%, and 5-year survival rate is about 50%.

Treatment of ischemic diseases include a number of measures of medical character, lifestyle changes, correction of comorbidity, however, the most pronounced effect of surgical and endovascular methods of recovery of disturbed blood flow - bypass surgery, angioplasty, atherectomy, stenting. Since conservative treatment methods are ineffective in a significant part of the heavy categories of these patients, and existing surgical and endovascular methods are applicable only for some patients, there is a fairly significant Sigorta patients with multiple distal stenosis of blood vessels, for which the only alternative is therapy of angiogenic growth factors, aimed at stimulating the growth of new blood vessels in the area of impaired blood flow (ischemia), which will allow without surgery to improve blood circulation and stop the degenerative processes, and thus to restore its function is tion. However, clinical trials of drugs based on recombinant growth factors or their genes for the treatment of coronary heart disease and critical ischaemia of the lower limbs did not show sufficient efficacy (Rissanen TT, Ylä-Herttuala S. Current status of cardiovascular gene therapy. Mol Ther. 2007; 15: 1233-47 // Gupta R, Losordo DW. Challenges in the translation of cardiovascular cell therapy. J Nucl Med. 2010 May 1; 51 Suppl 1:122S-127S. Review). The main reason for this is the low efficiency of transfection of tissue by direct introduction of genetic constructs and side effects associated with the use of some viral structures, which limits the possibility of re-introductions.

This necessitates the development of innovative methods of treatment of ischemic diseases, one of which is therapy stem and progenitor cells in the direction of regenerative medicine, is actively developing in recent years.

Numerous studies on models of chronic ischemia of the heart and myocardial infarction and ischemia of the extremities in animals have demonstrated the ability to restore blood supply to the heart, its structure and function after a heart using stem cells derived from various sources, including cells from bone marrow and adipose tissue, peripheral blood and cord blood, etc. it is known use in clinical practice of autologica the stem cells in an effective amount ranging from 10 6up to 1010cells to the patient (Stem cell treatment for acute myocardial infarction (Review). The Cochrane collaboration. Published by John Wiley&Sons, Ltd., published in The Cochrane Library 2012, Issue 2, http://www.thecochranelibrary.com).

However, in clinical studies the efficacy of cell therapy of ischemic diseases with autologous (from the patient) cells was not high enough (Kang S, et al. Effects of intracoronary autologous bone marrow cells on left ventricular function in acute myocardial infarction: a systematic review and meta-analysis for randomized controlled trials. Coron Artery Dis. 2008 Aug; 19 (5): 327-35. Review // Tongers J, Losordo DW, Landmesser U. Stem and progenitor cell-based therapy in ischaemic heart disease: promise, uncertainties, and challenges. Eur Heart J. 2011 May; 32 (10): 1197-206. Review.

One of the main reasons for the lack of effectiveness of autologous cell therapy is the reduction of the regenerative potential of stem cells under the influence of long-existing pathological process. Part of stem and progenitor cells in the recovery process of damaged tissues and organs at the expense of their functional features: the ability to directed migration in the damage zone, the differentiation into cells of blood vessels and the heart muscle and to the production of a wide range of biologically active molecules, growth factors, cytokines, enzymes. The latter property is considered to be the main mechanism of their regenerative action. Due to secretion (production) biologically AK is active molecules stem cells stimulate the growth of blood vessels and nerves in the ischemic tissue, help restore damaged cells and stimulate their own resident stem cells tissue. Violation of these functions leads to the decrease of the regenerative abilities of stem and progenitor cells for transplantation. Thus, endothelial progenitor cells (EPC) of bone marrow obtained from patients with coronary artery disease, produce less growth factors, much worse restore blood flow to ischemic limbs with the introduction of immunodeficient mice than EPA healthy people of the same age (Heeschen C. Et al. Profoundly Reduced Neovascularization Capacity of Bone Marrow Mononuclear Cells Derived From Patients With Chronic Ischemic Heart Disease. Circulation 2004; 109; 1615-1622).

Because the production of growth factors and cytokines stem cells when transplanted into the injured tissue is a key process that encourages the repair of tissue, strengthening these products due to the genetic modification can significantly improve the efficiency of transplantation of stem cells has been demonstrated in the work of Yu YS, et al. AKT-modified autologous intracoronary mesenchymal stem cells prevent remodeling and repair in infarcted swine myocardium. Clin Med J. 2010 Jul; 123 (13): 1702-8, in which modification of progenitor cells designs with the genes of intracellular signaling molecules Akt kinase that regulates proliferation and apoptosis of cells, leads to a significant improvement in their ability to recover miokar the animals after a heart attack.

Genetic modification of stem cells genes growth factors can solve the problem of insufficient efficiency of gene and cell therapy. Cells can be modified in vitro viral structures that will allow you to avoid direct introduction of these constructs into the fabric. The production of the target protein is a growth factor can be measured and the required number of cells can grow in culture. This gives the opportunity to measure the effect that it is practically impossible in case of direct introduction of genetic constructs. Selection of genes for modification of cells, so that their products had a complementary effect on the disease process, will enhance therapeutic effect through the use of a mixture of cell cultures, producing different growth factors. Key to the development of this technique is the selection of cells, the most suitable for genetic modification and transplantatio. These cells should be relatively easily obtained from the patient, should be modified safest viral structures and has for its properties to enhance the effects of those factors that they hyperproducers.

Today the greatest potential in regenerative medicine contact using multipotent mesenchymal stromal cells (MSCS) in bone mo the ha and adipose tissue. These cells are able to differentiate in vitro and in vivo in cells of blood vessels, stimulate the growth of blood vessels and nerves due to secretion of a wide range of growth factors, cytokines and proteases. These cells exhibit the properties of pericytes, contributing to stabilization of blood vessels. And MSC from adipose tissue, having the same properties as MSC from bone marrow, it is much easier to obtain due to the availability of adipose tissue in sufficient quantity in minimally invasive and painless procedure limited liposuction. All these properties of MSC from adipose tissue make them ideal targets for genetic modification to improve their secretory activity and their regenerative properties.

Because the key recovery process of the structure and function of tissues in ischemic damage is the restoration of blood flow due to the growth and remodeling of blood vessels, modification of various progenitor cells designs with the genome of the main factor angiogenesis - VEGF seems to be the most preferred for the treatment of ischemic tissue damage. However, there is data showing that overproduction of VEGF may lead to the development of tissue edema due to the formation of unstable immature "fluid" vessels and in some cases to the development of angioma (Carmeliet P. VEGF gene therapy: stimulating angiogenesis or angioma-genesis. Nat Md. 2000; 6. - 1102-3).

Vascularization (the growth of blood vessels) tissue is a complex multistep process involving many growth factors and cell types, and the formation of a Mature and functional vascular network is provided by the coordinated expression of different factors. So, when using direct gene therapy plasmid with the gene of platelet-derived growth factor (PDGF-BB) in combination with FGF-2 was able to obtain a significant increase in angiogenic effect and the formation of stable and functional blood vessels in comparison with using only plasmids with the gene FGF-2 (Cao R, Brakenhielm E, Pawliuk R, et al. The Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF. Nat Med 2003; 9: 604-13).

The closest to the technical nature of the claimed tool and method is use to stimulate vascularization and reparative processes in the myocardium after infarction using genetically modified bone marrow MSC, do overexpress VEGF, a gene which is under hypoxic promoter, that is expressed only under conditions of hypoxia (Kim SH, et al. Hypoxia-inducible Vascular Endothelial Growth Factor-engineered Mesenchymal Stem Cells Prevent Myocardial Ischemic Injury. Mol Ther. 2011 Jan 18.) It was stimulirovalis the growth of blood vessels and reduced the size of infarction (necrosis of the myocardium). However, obtaining cells from the bone marrow is associated with painful and significantly more burdensome for the Aulnay procedure puncture of the iliac bones, what procedure is limited liposuction, which is used to obtain MSC from adipose tissue. In addition, a significant increase in VEGF production in the microenvironment of the transplanted genetically modified cells can lead to the preferential development of unstable immature and functionally inactive vessels that are subjected to reverse development, when the concentration of VEGF in the tissue is reduced (Ozawa CR, Banfi A, Glazer NL, et al. Microenvironmental VEGF concentration, not total dose, determines a threshold between normal and by aberrant angiogenesis. J Clin Invest 2004; 113: 516-27). This leads to a short temporary effect of therapy based on the increase in production of only one VEGF in chemisorbing tissues (Kholova I, Koota S, Kaskenpää N, et al. Adenovirus-mediated gene transfer of human vascular endothelial growth factor-d dosage transient angiogenic effects in mouse hind limb muscle. Hum Gene Ther. 2007 Mar; 18 (3): 232-44), and can also cause the development of tissue edema, as VEGF is the most powerful factor in increasing vascular permeability (Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced vascular permeability. Nature. 2005 Sep 22; 437 (7058): 497-504). All this requires the use together with VEGF factors stabilizing the vessel, reducing vascular permeability and attract pericyte, smooth-muscle cells, forming the muscular layer of the vascular wall, and fibroblasts. Therefore, the vessels remain stable after the concentration of VEGF in the tissue, reduces energy is camping, that provides lasting effect of vascularization, leading to the restoration of the structure and function of tissues. These factors include angiopoietin-1, platelet-derived growth factor-BB (PDFG-BB), fibroblast growth factors (FGF), transforming growth factor-beta (TGF-beta). All of these factors in its action helps stabilize blood vessels, however, not obvious is that by increasing their production in ischemic tissues with increased VEGF production will be stimulated as the growth of blood vessels and their stabilization, as some of these factors under certain conditions, is able to inhibit angiogenesis.

The objective of the invention is to develop a new means and method of treating ischemic lesions of tissues.

The technical result is to accelerate vascularization and repair tissue by creating a combination of genetically modified cells, hyperproducers two growth factor, a joint action which leads to the formation of a Mature and functional vascular network, providing a more effective restoration of disturbed blood flow and tissue structure than the use of direct injection of unmodified cells or modified cells, hyperproducers one growth factor.

The problem is solved in that the treatment for Isha is practical lesion tissue is a mixture of the two cultures of mesenchymal stem cells, one of which is modified by genetic design bearing the gene growth factor vascular endothelial (hVEGF), and the other genetic design bearing the gene of angiopoietin - 1 (ANGPT-1). As genetic designs can be used recombinant adeno-associated viral vector or adenoviral or lentiviral transfer vector, and cells - autologous or allogeneic cells from adipose tissue or bone marrow. The components of the mixture taken in the ratio 1÷0.5 to 3. The optimal result is achieved when the components of the mixture taken in the ratio 1÷1.

The task is also solved by the fact that the method of treatment of ischemic lesions tissue includes introducing a therapeutically acceptable amount in ishemizirovanne fabric means, diluted in a solution of a therapeutic culture medium not containing serum. When this product is used in a concentration of from 3 to 100 million cells in 1 ml medium by injections ischemic tissue.

Studies have shown that the best result is achieved by using a mixture of cultures modified mesenchymal stem (stromal) cells, hyperproducers angiogenic growth factors VEGF and ANGPT-1 ratio 1:1, 1:2, 1:3, 2:1 and 3:1.

A method of treating ischemic lesions of the tissues is the introduction way of the multiple injections (injections) itemizedoverlay fabric, for example the muscles of the limbs or the myocardium, in a culture medium not containing serum, the mixture of cultures modified mesenchymal stem (stromal) cells, hyperproducers VEGF and ANGPT-1 in concentrations of from 3 to 100 million cells in 1 ml of solution, the number of points for injection and used the number of cells depends on the size of the ischemic region of tissue.

Genetically modified mesenchymal stromal cells from adipose tissue were obtained using the transformation of these cells with recombinant adeno-associated viral vector serotype 2 - AAV Helper-Free System (Stratagene, USA), which were integrated genes VEGF and ANGPT-1 person.

Comprehensive feature was obtained by mixing cultures of cells that produce VEGF and ANGPT-1, the ratio of the number of cells 1:1, 1:2, 1:3, 3:1, 2:1 and breeding in a solution of sterile therapeutic environment for mesenchymal cells to a concentration of from 1 to 100 million cells per ml

As a culture medium in which the cells will be introduced into the tissue, can be used commercially available therapeutic environment that does not contain serum, such as AIM V® Serum Free Medium, INVITROGEN, USA; line therapeutic media company PAA_Therapeutics, USA; Advance STEM, HyClone, USA.

The invention is illustrated by drawings.

Figure 1 shows the increase in production of VEGF and ANGPT-1 modified cells, assessed with the apprehension of VEGF-165 and ANGPT-1 in air-conditioned environment intact MSC from adipose tissue (adipose tissue MSC), MSC-modified virus with the gene marker protein GFP (GFP-MCK VT) and MSC-modified virus with a genome of VEGF-165 (VEGF-MCK VT) (A) or genome ANGPT-1 (ANGPT-1 - MSC in adipose tissue) (B) (n=5). Data show that in the modified cells the production of the target protein is a growth factor increases tenfold.

Figure 2 shows the number of capillaries and arterioles (B) - Mature vessels with smooth muscle layer of Matrigel implants under the skin of the mouse in the case of the introduction: 1) of Matrigel with non-modified cells (MSCS VT); 2) the Matrigel cells, hyperproducers VEGF (VEGF-MCK adipose tissue); 3) the Matrigel cells, hyperproducers ANGPT-1 (ANGPT-1 - MSC VT); 4) of Matrigel with a mixture of cells, hyperproducers ANGPT-1 and VEGF (VEGF-MCK VT+ANGPT-1 - MSC in adipose tissue). From figure 2 it is seen that the introduction of a subcutaneous implant of Matrigel mixture of cells, hyperproducers VEGF and ANGPT-1 leads to sprouting in Matrigel greatest number of vessels like capillaries and muscle membrane (stable vessels) compared with the introduction of unmodified cells, hyperproducers one of the factors (p<0.01), and that is the greatest vascularization of Matrigel implant is achieved through a mixture of cells that produce VEGF and ANGPT 1.

Figure 3 shows the number of capillaries and arterioles (B) in the muscles of the ischemic hind limb of mice after administration of: 1) unmodified cells(MSCS VT); 2) cells, hyperproducers VEGF (VEGF-MCK adipose tissue); 3) cells, hyperproducers ANGPT-1 (ANGPT-1 - MSC VT); 4) a mixture of equal amounts of cells, hyperproducers ANGPT-1 and VEGF (VEGF-MCK VT+ANGPT-1 - MSC in adipose tissue). From figure 3 it is seen that the intramuscular transplantation of a mixture of cells, hyperproducers VEGF and ANGPT-1, leads to the formation of more stable vessels with muscular sheath (p<0.05)than transplantation of unmodified cells or cells that produce one of these factors.

Figure 4 shows the decrease of the area of necrosis, reflecting the reparative process in ishemizirovannyh the muscles of the hind limb of the mouse, after the introduction of: 1) unmodified cells (MSCS VT); 2) cells, hyperproducers VEGF (VEGF-MSC in adipose tissue); 3) cells, hyperproducers ANGPT-1 (ANGPT-1 - MSC VT); 4) a mixture of equal amounts of cells, hyperproducers ANGPT-1 and VEGF (VEGF-MCK VT+ANGPT-1 - MSC in adipose tissue). From figure 4 it is seen that the most pronounced decrease the zone of necrosis in the ischemic muscle of the hind limbs was observed in the transplantation of a mixture of cells, hyperproducers ANGPT-1 and VEGF (p<0,02). Increase the effectiveness of restorative processes is achieved through the use of a mixture of modified cells that produce ANGPT-1 and VEGF.

The methodology of the experiments and the results obtained are illustrated by the following examples.

Example 1. Obtaining genetically Modific the level of cells and evaluation of the products of their growth factors. To obtain the MSC human adipose issue, do overexpress VEGF and angiopoietin-1 were created appropriate adeno-associated viral vectors encoding these genes, for which we used vector system for gene transfer based on adeno-associated virus 2 on serotype - AAV Helper-Free System (Stratagene, USA). Production of recombinant adeno-associated viruses is carried out in the cell line SOME T, cotranslationally the plasmid AAV-RC (encodes the viral genes necessary for replication and encapsidation virus), pHelper (encodes a gene of adenovirus) and vector plasmid pAAV-VEGF or pAAV-ANGPT-1, respectively. To obtain vector plasmids used optimized gene VEGF 165 person and optimized gene ANGPT-1.

To obtain virus encoding the marker gene green fluorescent protein - GFP, cells were cotranslational plasmids pAAV-RC, pHelper and pAAV-hrGFP (Stratagene). To obtain virus carrying the gene for VEGF 165 human or gene angiopoietin-1 person, instead of the plasmid pAAV-hrGFP used cloned plasmids pAAV-hVEGF and pAAV-hAng-1. The transfection was carried out using the calcium phosphate method.

Human MSC were isolated from the material intact subcutaneous adipose tissue, cut off at surgery from patients older than 18 years and modified by the authors of the method of Separation et al. (Separation, P.A., Zhu, M., Ashjian, P., et al. (2002) Human adipose tissue isa source of multipotent stem cells. Mol. Biol. Cell. 13, 4279-4295).

For all experiments the use of MSC in adipose tissue is not higher than 4 passages. For modification of MSC in adipose tissue recombinant adeno-associated virus cells 1-2 passage cultivate in the environment AdvanceSTEM/10%GS. When the cells 60-70% confluent monolayer environment replace AdvanceSTEM/1%GS cooled to 4°C, and add viral drug at the rate of 1-2 ml of virus preparation at 400-600 thousand cells (one Petri dish with a diameter of 100 mm). Incubate the cells for 4 hours at 37°C in CO2-incubator, stirring Wednesday every 30 minutes Next to FIA GT in the environment AdvanceSTEM/1%GS/viral drug add an equal volume of medium AdvanceSTEM/20%GS and cultured cells without changing the environment.

The effectiveness of modification of cells with constructs carrying the genes VEGF and ANGPT-1, was estimated by the increase in air-conditioned environment of the product of the transgene compared with its content in the environment, air conditioned, non-modified cells. Quantitative analysis of the accumulation of the products of the transgenes (VEGF and angiopoietin-1) in air-conditioned environments, conducting enzyme-linked immunosorbent assay using the appropriate reagent kit for Human VEGF Quantikine Kit (R&D Systems, USA) and Human Angiopoetin-1 Quantikine Kit (R&D Systems, USA) in strict accordance with the manufacturers Protocol. When effective modification of the MSC in adipose tissue viral structures increase the product of the transgene in the schools is focused environment should no less than 10 times higher than its content in air-conditioned environment unmodified cells (figure 1).

Example 2. Analysis of vascularization subcutaneous implant of Matrigel (model subcutaneous angiogenesis). In this work, we used the male immunodeficient mice of BALB/c NUDE (Nude mice with deficiency of T-cells) at the age of 8-10 weeks. All manipulations with animals were performed in accordance with the internal Rules of work with the use of experimental animals"approved by the fgbi "Russian cardiological center" health Ministry in a special clean room under the kelp. For the experiment, the mice of BALB/c NUDE subcutaneously in the region of the groin was performed with the injection of 400 μl of Matrigel (Matrigel, BD Biosciences, USA), mixed with 100 μl of the FSB or 100 µl of cell suspension (5×105cells). 14 days after the beginning of the experiment were removed implants of matrimelee, was placed in the cuvette with the environment OCT Tissue-Tek (Sakura, Japan) and frozen in liquid nitrogen.

Samples of the implants of matrimelee was frozen in liquid nitrogen in the environment OCT Tissue-Tek (Sakura, Japan). The samples were stored at a temperature of 70°C. Histological analysis was performed on frozen sections with a thickness of 7-10 μm. For visualization of blood vessels growing in Matrigel, used staining with antibodies against smooth muscle actin mouse (SMA) (FITC-SMA, 1:100, Sigma-Aldrich, USA) for visualization of vessels with muscular skin and unlabeled rat antibody against mouse CD31 (anti-CD31, 1:100, BD Biosciences, USA) for visualiz is of all vessels.

The sections were analyzed under a microscope Axiovert 200M (Zeiss, Germany). Document images and their subsequent processing was made using a digital camera Axiocam HRC (Zeiss, Germany) software Axiovision 3.1 (Zeiss, Germany), MetaMorph 7.1.0.0 (Universal Imaging Corporation, USA). For each slice samples of Matrigel using the digital camera has received a series of random shots, covering more than 80% of the cut surface, which conducted morphometrically research followed by statistical processing of the data.

The results are shown in figure 2.

Example 3. Model of ischemia of the hind limb of the mouse. To assess therapeutic potential of MSC in adipose tissue, hypersecretory growth factors and cytokines, used a model of ischemia of the hind limb of the mouse, as described earlier in Couffinhal T. et al. (Couffinhal T., Silver, M., Zheng L.P., et al. Mouse model of angiogenesis. Am J Pathol. 1998; 152 (6): 1667-79), developed for mice C57b1. In our experiments we used a line of mice BALB/c Nude deemed acceptable to study the angiogenic potential of human cells during transplantation. However, this line of mice is characterized by poor development of collateral vessels in comparison with the line C57b1 (Mac Gabhann f, S.M. Peirce Collateral capillary arterialization following arteriolar ligation in murine skeletal muscle. Microcirculation. 2010; 17 (5): 333-47; Chalothom D., J.E. Faber Strain-dependent variation in collateral circulatory function in mouse hindlimb. Physiol Genomis. 2010; 42 (3): 469-79), so induced by excision of the femoral artery ischemia in BALB/c mice leads to extensive tissue necrosis and loss of limb. In this regard, the model was modified by us to BALB/c mice by replacing excision a.Femoralis it ligation, allowing efficient enough to reduce blood flow in the limb, but does not lead to high amputations, and therefore it has become possible to assess the proliferation of blood vessels and the proportion of muscle necrosis. Mice were injected anaesthesia using intraperitoneally injection of 300 μl of a 2.5% solution of Avertin. The operation was performed under a stereomicroscope Olympus SZX16 (Olympus, Japan). In the left femoral region of the mouse performed the section on projection a.femoralis. The femoral artery was isolated throughout its distal part and tied with silk thread 6-0 proximal point of the popliteal bifurcation preserving intact v.femoralis and n.ischiadicus. Wound sutured atraumatic needle (4-0 silk) continuous suture, and the skin was sterilized with 70% ethanol and placed the animal in a separate cage until full recovery from anesthesia.

Cell culture resuspendable in 0.15 ml of phosphate-saline buffer or serum-free culture medium was injected in an amount of 5×105cells at the animal 3 points in ishemizirovanne the muscles of the limb m.tibialis anterior, m.gastrocnemius and m.biceps femoris.

Samples of muscle were isolated on day 21 after surgery and samarajiva liquid nitrogen in the environment ACT Tissue-Tek (Sakura, Japan). The samples were stored at a temperature of - 70C. Histological analysis was performed on frozen sections with a thickness of 7-10 μm. Sections of muscle tissue for visualization of vessels using primary-labeled antibodies against smooth muscle actin mouse (SMA) (FITC-SMA, 1:100, Sigma-Aldrich, USA) and unlabeled rat antibody against mouse CD31 (anti-CD31, 1:100, BD Biosciences, USA). Cell nuclei were stained with nuclear dye DAPI for 10 min, after which the preparations were washed with buffer FSB, concluded on Wednesday Aqua polymount (Polysciences inc., USA) and covered with cover glass. To visualize the area of necrosis of the muscle tissue slices were stained with hematoxylin-eosin.

The sections were analyzed under a microscope Axiovert 200M (Zeiss, Germany). Document images and their subsequent processing was made using a digital camera Axiocam HRC (Zeiss, Germany) software Axiovision 3.1 (Zeiss, Germany), MetaMorph 7.1.0.0 (Universal Imaging Corporation, USA). For each slice m.tibialis anterior using the digital camera has received a series of random shots, covering more than 80% of the cut surface, which conducted a morphometric study followed by statistical processing of the data.

The results presented in figure 3 and figure 4 show that the introduction ishemizirovanne the muscles of the hind limb of the mouse a mixture of cells that produce VEGF and ANGPT-1, allows to reach l is CSA vascularization of ischemic tissues due to a larger number of generated stable vessels, having a muscle layer than the introduction of unmodified cells or cells that produce one of these factors. When this occurs, the most significant decrease in necrosis of the muscles, indicating a more pronounced reduction processes.

The data presented in the format of mean ± standard deviation. Statistical processing of results was performed using the Statsoft Statistica 6.0. Used t-student test or U-test Mann-Whitney for samples with Gaussian or non-parametric distribution of values, respectively. Significant believed differences with p<0,05.

1. For the treatment of ischemic tissue lesions, representing a mixture of the two cultures of mesenchymal stem cells, one of which is modified by genetic design based viral vector that provides the overproduction of growth factors, vascular endothelial, and the other modified genetic structure based viral vector that provides the over production of angiopoietin, the components of the mixture taken in the ratio 1÷0.5 to 3 in an effective amount.

2. The tool according to claim 1, characterized in that as the cells used autologous or allogeneic mesenchymal stem cells from adipose tissue or bone marrow.

3. The tool according to claim 1, harakteryzuyetsya, what components of the mixture taken in the ratio 1÷1.

4. A method of treating ischemic lesions of tissues, including the introduction of a therapeutically acceptable amount in ishemizirovanne fabric means according to claim 1 dissolved in a solution of therapeutic culture medium not containing serum of animals, while the product is used in a concentration of from 3 to 100 million cells in 1 ml of culture medium through injections ischemic tissue.



 

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FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of immunology. Claimed is a version of Fc polypeptide of human IgG with substitutions 2591 and 308F, where numeration of positions is given in accordance with EU Kabat index. Described is a version of the said polypeptide, including one or several substitutions of the following: 428L, 434S, 307Q, 319L, 250I in addition to the said ones. Disclosed are: a nucleic acid, coding the said versions, a host cell for production of the said versions of polypeptide, which contains the coding nucleic acid, a method of obtaining the said versions of polypeptide, including application of the cell expressing the said polypeptide and containing the nucleic acid, which codes the said polypeptide.

EFFECT: application of the invention provides polypeptide, demonstrating higher affinity with human FcRn, which can be applied in therapy of different diseases.

11 cl, 32 dwg, 14 ex

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biology and chemistry and deals with isolated nucleic acid, coding fluorescent protein with biosensor properties, expression cassettes, providing expression of said fluorescent protein, cells, producing said protein, and peculiarly fluorescent protein with biosensor properties. Obtained fluorescent protein has amino acid sequence, given in SEQ ID NO:4, and intended for changing NAD+/NADH ratio inside cells by increasing signal with displacement of NAD+/NADH ratio towards decrease of NADH concentration.

EFFECT: claimed invention makes it possible to carry out analysis of processes in cell in real time mode.

4 cl, 6 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology. Described is molecule of chimeric nucleic acid of porcine circovirus (PCV2Gen-1Rep), which includes molecule of nucleic acid, coding porcine circovirus of type II (PCV2), which contains sequence of nucleic acid, coding protein Rep of porcine circovirus of type 1 (PCV1). Chimeric molecule of nucleic acid is constructed by replacement of gene Rep ORF1 PCV2 with gene Rep ORF1 PCV1. Invention also includes biologically functional plasmid or viral vector, which contain unique molecules of chimeric nucleic acids, suitable host cells, transformed by plasmid or vector, infectious chimeric porcine circoviruses, which produce suitable host cells, method of obtaining immunogenic polypeptide product with application of novel chimera, viral vaccines, protecting pig against viral infection or syndrome of postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of protecting pigs against viral infection or postweaning multisystem wasting syndrome (PMWS), caused by PCV2, methods of obtaining unique chimera PCV2Gen-1Rep and the like. Invention can be applied in veterinary.

EFFECT: invention additionally includes novel method of increasing level of replication and PCV2 titre in cell culture.

21 cl, 2 dwg, 6 ex

Fused rage proteins // 2513695

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biochemistry. Claimed is fused protein for treating diseases, mediated by advanced glycation end products (AGE), consisting of a fragment of a version of human receptor of advanced glycation end products (RAGE), which has two point mutations H217R and R221H, and a fragment of constant domain of human immunoglobulin IgG4, joined with linker if necessary. In addition, considered are: nucleic acid and recombinant host cell for obtaining fused protein, as well as pharmaceutical composition for treatment of AGE-mediated diseases, which contain fused protein.

EFFECT: invention ensures lower aggregation of fused protein.

13 cl, 19 dwg, 3 ex, 9 tbl

FIELD: chemistry.

SUBSTANCE: claimed invention relates to field of biotechnology, in particular to novel peptide analogue of insulin-like growth factor-1 (IGF-1), which contains amino acid substitution of methionine in position 59 on Asn, Leu, Nle, Ile, Arg, A6c, Glu, Trp or Tyr, as well as other additional substitutions, inserts and deletions. Said peptide or its pharmaceutically acceptable salt is used in composition of pharmaceutical composition for treatment of IGF-1-mediated diseases, as well as in method of treating dwarfism.

EFFECT: invention makes it possible to obtain IGF-1 analogue-agonist, possessing higher biological activity with respect to native IGF-1.

17 cl, 2 tbl

FIELD: medicine.

SUBSTANCE: present group of inventions relates to biotechnology. What is presented is a humanised anti-CD79b antibody and its antigen-binding fragment produced of murine antibody MA79b and CD79b having a substantially analogous binding affinity thereto. A polynucleotide, a vector, a host cell and a method for producing the anti-CD79b antibody according to the invention; immunoconjugates, compositions and methods for cell growth inhibition, a method of treating an individual suffering cancer, a method of treating a proliferative disease and tumour in a mammal, a method for B-cell proliferation inhibition; a method for detecting the presence of CD79b in a sample and method for binding the antibody to the CD79b expressing cell are also disclosed.

EFFECT: given invention can find further application in therapy of the CD79b associated diseases.

86 cl, 20 tbl, 9 ex, 51 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology. A method includes stages of cultivating cells, expressing markers, characteristic of a line of pancreatic endocrine cells, in a medium, which contains a sufficient amount of a cyclin-dependent kinase inhibitor to induce increase of MAFA expression. As the cyclin-dependent kinase inhibitor used is ethyl-(6-hydroxy-4-phenylbenzo[4,5]furo[2,3-b])pyridine-carboxylate. The invention can be used in medicine.

EFFECT: claimed is the method of increasing MAFA expression in cells, expressing the markers characteristic of the line of the pancreatic endocrine cells.

3 cl, 10 dwg, 7 tbl, 9 ex

FIELD: biotechnology.

SUBSTANCE: method comprises isolation of mononuclear cells (MNC) from peripheral blood of a patient, separation of cells to adherent and non-adherent fractions, addition of the adherent fraction to the MNC of growth factors, loading of the dendritic cell with antigens of tumour lysate in vitro, the stimulation of maturation of dendritic cells for the next day. At that, the obtained immature DCs are added to lysate-autologous tumour cells at a dose of 100 mcg/ml, and after 48 hours within the subsequent 24 hours the rf-tumour necrosis factor-alpha is applied at a dose of 25 ng/ml. Then, the co-culture is carried out of mature dendritic cells activated with lysate and the non-adherent fraction of MNC at a ratio of 1:10 in the presence of recombinant human interleukin-12 at a dose of 10 ng/ml and the recombinant human interleukin-18 at a dose of 100 ng/ml.

EFFECT: invention enables to improve the level of cytotoxic and interferon-producing activity of antigen-activated dendritic cells while reducing the duration of their culture.

4 tbl

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to dentistry, and may be used for making a restoration material used for partial dental restoration in the oral cavity. That is ensured by placing the first cell mass formed by mesenchymal or epithelial cells/cell and a second cell mass formed by other mesenchymal or epithelial cells/cell on a carrier. One of the mesenchymal or epithelial cells is made from a dental germ, and the above cell masses are placed in tight contact to each other, but not mixed. The above cell masses are grown to form the whole restored tooth or its germ. That is followed by localising the whole restored tooth or its germ grown that enables implanting the whole restored tooth or is germ within the lost tooth so that a dental crown is directed inside the oral cavity with the dental germ or the tooth being used as the restoration material for preparing an equivalent of the lost tooth within the lost tooth area.

EFFECT: group of inventions enables performing partial dental restoration within the lost tooth area by implanting the restored dental germ or the whole restored tooth, as prepared by the above method.

10 cl, 5 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: method comprises euthanasia of infant rabbits by decapitation, removing paws, tails, removing skins, opening the abdomen cavity at the level of the lumbar vertebrae, kidney extraction, removal of the organ capsule, shredding into pieces with the size of 1-3 mm. The donors of the primary cell culture the newborn rabbits 1-2 days old are used. The pieces of tissue are washed from residual blood with saline solution of Hanks, subjected to disaggregation in 0.25% trypsin solution by preliminary incubation of the pieces at 37°C for 30-40 minutes, the cells are pelleted by centrifugation at 1000-1500 rev/min for 10 minutes, the supernatant is drained off, and the pellet is resuspended in the growth medium consisting of medium Igla MEM, 0.5% solution of lactalbumin (1:1) with the pH 7.0-7.2 with 10% serum of adult cattle or foetuses of cows, the cell concentration is adjusted to 5-7·105 cells/cm3, then antibiotic ciprofloxacin at 100 U/ml is added. At that the sensitivity of primarily trypsinised cultures of kidney cells of newborn rabbits to respiratory-enteric viruses of cattle, parvoviruses, herpesviruses and parainfluenza-3 by determining the viral titers in lg 50/ml TDC is determined.

EFFECT: method enables to enhance the sensitivity to viruses of animals, to improve the biological activity of the cultured cells, and to increase the yield of viral material.

3 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and represents a method of obtaining a polypeptide in a culture of mammalian cells (versions) and a medium for cultivation of the mammalian cells. The claimed invention can be used for obtaining the polypeptide of interest in great amounts. The method includes obtaining the cell culture, which contains the mammalian cells containing a nucleic acid, which codes the polypeptide of interest, and an initial medium for cell cultivation, with a volume of the initial medium for cell cultivation constituting approximately 60-99% of a volume of the desired cell cultivation medium. The nutritional medium for cultivation is added to the cell culture, with a volume of the nutritional medium for cell cultivation constituting approximately 1-40% of a volume of the cell cultivation medium. The obtained medium for cell cultivation represents a medium for cell cultivation, containing from approximately 7 mM to approximately 30 mM of leucine, from approximately 7 mM to approximately 30 mM of lysine, from approximately 7 mM to approximately 30 mM of threonine, from approximately 7 mM to approximately 30 mM of proline and from approximately 7 mM to approximately 30 mM of valine. The cell culture is kept under conditions, which allow expressing the polypeptide of interest.

EFFECT: claimed invention makes it possible to obtain the polypeptide of interest with a higher output.

18 cl, 15 dwg, 16 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to immunology, medicine and biotechnology. What is presented is a method for stimulating a specific anti-tumour immune response to breast cancer cells with the use of dendritic cells transfected by polyepitope DNA construct. The cells recovered from the peripheral blood of relatively healthy donors, transfected by the polyepitope structure are cultured with a non-adherent fraction of mononuclear cells.

EFFECT: method enables providing higher effectiveness and cost effectiveness of preparing cytotoxic lymphocytes.

1 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to medicine and veterinary science. What is presented is a biocomposite for performing reparative processes following an injury in a mammal, containing a carrier, at least one nucleic acid containing genes coding VEGF and/or SDF-1, and cells ensuring reparative regeneration. There are presented methods for preparing the above biocomposite and a kit for preparing the same. There are also presented a method for providing injury healing in a mammal and a method for delivering nucleic acid.

EFFECT: presented group of inventions provides effective tissue regeneration following the injury in the mammal by using the three-component biocomposite consisting of the carrier, at least one nucleic acid and the cells ensuring reparative regeneration.

16 cl, 4 dwg, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to preparing recombinant proteins and concerns methods for preparing them. There are presented methods for preparing antibodies, involving: culturing mammalian cells containing nucleic acid coding an antibody or its fragment in a cell culture containing a production medium of the cell cultures; feeding the mammalian cells by adding a hydrolysate-rich solution and a rich solution of a basal medium to the cell culture, wherein the hydrolysate-rich solution contains a herbal hydrolysate and a yeast hydrolysate 75-300 g/ml. The presented methods can be used to prepare particularly such antibodies, as anti-TNFα-antibody or anti-IL-12-antibody.

EFFECT: characterised solution enables preparing high antibody count and may be used in pharmaceutical industry.

28 cl, 46 tbl, 4 ex, 1 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry, particularly to antibodies specifically bound to epidermal growth factor receptors (EGFR), as well as to DNA coding V heavy chain regions of the above antibodies, to DNA coding V light chain regions of the above antibodies. There are disclosed expression vectors containing these DNAs, and animal cell lines for expression of the above antibodies containing these vectors. There are described compositions for treating cancer related to epidermal growth factor receptor (EGFR) containing an effective amount of the above antibodies.

EFFECT: invention enables treating cancer related to epidermal growth factor receptor (EGFR) effectively.

30 cl, 12 dwg, 12 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: invention relates to the field of cellular biology and biotechnology, as well as to medicine. Claimed is a method of cultivating mesenchymal stem cells, isolated from the bone marrow.

EFFECT: due to high homogeneity and efficiency of isolation of stem cells the method can be used in transfusiology.

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to new phenylpyrimidone derivatives of formula I possessing the properties of a phosphodiesterase type 5 (PDE5) inhibitor. The compounds of formula I can be used for treating various vascular disorders, such as erectile dysfunction, pulmonary arterial hypertension, etc. In formula each R1 and R2 independently means H; C1-C10alkyl; halogen; CF3; CN; OR5; NR6R7; NHCOR8; aryl; or C1-C4alkyl optionally substituted by OR5; Z means OR3; R3 means C1-C6alkyl or C1-C3alkyl, substituted by C1-C3alkoxy group; R4 means SO2NR6R7; NR9R10, providing NR9R10 is other than NH2; COR11; OR12; or R4 means 5-6-merous heterocyclyl optionally substituted by one or more substitutes specified in a group consisting of OH and C1-C6 alkyl; or R4 means 5- or 6-merous cyclic monosaccharide group; R5 means C1-C6alkyl; C1-C4alkyl optionally substituted by C1-C4alkoxy group; each R6 and R7 independently means H, OH, C1-C6alkyl, C1-C6alkoxy group, C3-C6alkenyl, C3-C6cycloalkyl, adamantyl, C3-C8lactamyl, aryl, Het or (CH2CH2O)jH, wherein j is 1-3; or each R6 and R7 independently means C1-C6alkyl, optionally substituted by OH, C1-C4alkoxy group, SO3H, SO2NR13R14, SO2R16, NR13R14, aryl, Het or 5-6-merous heterocyclyl; or each R6 and R7 independently means 5-6-merous heterocyclyl optionally substituted by one or more substitutes specified in a group consisting of C1-C6 alkyl and C1-C6alkyl substituted by hydroxyl; or R6 and R7 together with a nitrogen atom attached whereto form 5-7-merous heterocyclyl optionally substituted by one or more substitutes specified in a group consisting of OH, COOR8, (CH2CH2O)jH, wherein j is 1-3, C1-C4alkoxy group, Het and C1-C6alkyl substituted by aryl; or R6 and R7 together with a nitrogen atom attached whereto form a glucosyl amino group, an amino acid residue, a residue of an amino acid ester or an amino amide residue. The other radical values are specified in the patent claim.

EFFECT: invention refers to pharmaceutical compositions based on the above compounds, using them, methods for preparing the compounds, and intermediate products.

18 cl, 2 tbl, 224 ex

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