Method of estimating angiogenic potential of progenotor cells in patients with cardiovascular diseases

FIELD: medicine.

SUBSTANCE: claimed invention relates to the field of biotechnology, namely to the preliminary estimation of the efficiency of the autologic cell material transplantation to stimulate the growth of blood vessels, and can be applied in medicine. Claimed is a method of the complex estimation of the angiogenic potential of progenitor cells in patients with cardiovascular diseases, tested on mesenchymal stromal cells of the adipose tissue (MSC-AT) of patients with ischemic heart disease and including the measurement of content of mRNA and proteins of basic angiogenic factors, produced by the progenitor cells such as the vascular endothelial growth factor (VEGF), the placental growth factor (PIGF), the hepatocyte growth factor (HGF), angiopoetin-1 and angiogenin, the angiogenic activity of total cell secretion products, as well as the estimation of the ability of the progenitor cells to stimulate the vascularisation of subcutaneous Matrigel implants, introduced to immunodeficient mice. As the screening method used is a simpler and more available but less informative method of express-assessment of the angiogenic properties of the progenitor cells, based on the measurement of the angiogenic activity of the total cell secretion products.

EFFECT: invention makes it possible to carry out testing of the autologic cell material obtained from the patients, including those with ischemic heart disease, before transplantation in order to choose the optimal tactics of cell therapy aimed at the stimulation of the growth of blood vessels.

2 cl, 2 dwg, 4 tbl, 4 ex

 

The technical field to which the invention relates

The present invention relates to the field of cell biology, in particular cell Transplantology and tissue engineering, and can be used to pre-assess the effectiveness of autologous cell material to stimulate the growth of blood vessels that is necessary for the treatment of several human diseases, such as cardiovascular disease, damage to the nervous system, urological diseases, defects of the osseous system, diseases of the joints, periodontal disease, burns, and also for correction of age changes and skin defects in dermatology. A method of integrated assessment of angiogenic potential of progenitor cells in patients with cardiovascular disease, which has been tested on mesenchymal stromal cells of adipose tissue (MSC in adipose tissue in patients with coronary artery disease and includes the measurement of the content of mRNA and proteins of the major angiogenic factors produced by progenitor cells, angiogenic activity of total products of secretion cells, as well as evaluating the ability of progenitor cells to stimulate vascularization of subcutaneous implants of Matrigel entered immunodeficient mice. As a screening method can be ispolzovanie simple and affordable, but less accurate method of rapid assessment of the angiogenic properties of progenitor cells based on the measurement of angiogenic activity of total products of secretion. The invention allows the testing of autologous cell material obtained from patients, including coronary heart disease, prior to transplantation to choose the best course of cell therapy to stimulate the growth of blood vessels.

The level of technology

Cardiovascular diseases, including coronary heart disease (CHD), take first place in the structure of causes of death in most countries, despite considerable progress in the development of medical treatment and surgical and endovascular revascularization. One of the promising approaches to the treatment of therapeutic angiogenesis is based on the introduction to ishemizirovanne genetic tissue constructs with genes of growth factors or stem/progenitor cells. Many types of progenitor cells of the adult organism, including multipotent mesenchymal stromal cells isolated from bone marrow or adipose tissue (MSC in adipose tissue), can serve as a promising tool for therapeutic angiogenesis through its ability to stimulate the growth of blood vessels, in particular by sacreligion growth factors. As an example for the development of this invention were used MSC in adipose tissue of patients with coronary heart disease, because they are much easier to obtain in large enough quantities in minimally invasive procedure, limited liposuction or biopsy of adipose tissue, however, the disclosed invention may be applicable for any other types of progenitor cells with a similar mechanism of action, including stimulation of angiogenesis in the damaged tissues.

In models of limb ischemia and infarction in animals have shown that local and systemic administration of MSC in adipose tissue increases the number of blood vessels in tissues with impaired circulation and improve tissue perfusion with blood (Traktuev, 2006; Lee, 2006; Rehman, 2004; Miyahara, 2006; Nakagami, 2006; Gimble, 2007; Cai, 2009; Kondo, 2009). Restoration of blood flow in ischemic tissue in transplantation of MSC in adipose tissue caused by several mechanisms. First, these cells secrete a wide range of angiogenic growth factors that promote migration and proliferation of endothelial cells and their precursors, as well as the formation of new blood vessels (Rehman, 2004; Gimble, 2007; Kondo, 2009; Rubina, 2009; Madonna, 2010). Secondly, MSC in adipose tissue secrete plasminogen activators and matrix protease that contributes to local destruction of extracellular matrix and migration of cells involved in the formation of the vascular wall, and �also the release associated with the matrix of angiogenic factors (Kachgal, 2011). Thirdly, MSC in adipose tissue can differentiate into smooth muscle and endothelial cells embedded in the growing vessels, and stabilize newly formed vessels, performing the function of pericytes (Miranville, 2004; Planat-Benard, 2004; Miyahara, 2006; Sumi, 2007).

Thus, MSC in adipose tissue are the perfect material for cell therapy of ischemic diseases and are already used in early phases of several clinical trials (Madonna, 2009; Murohara, 2009; Bailey, 2010; Gimble, 2011). However, the vast majority of findings on angiogenic and regenerative properties of MSC in adipose tissue of man, obtained on cells isolated from adipose tissue relative to healthy young donors. At the same time, it is known that aging and the disease itself may have a negative impact on the MSK (Fehrer, 2005; Sethe, 2006; Stolzing, 2008; Katsara, 2011; Madonna, 2011; Sun, 2011). It is shown that during aging reduces proliferative capacity of MSC in adipose tissue and their ability to differentiation into adipogenic and osteogenic directions (Zhu, 2009; Huang, 2010), as well as worsen their angiogenic properties (El-Ftesi, 2009; Huang, 2010; Efimenko, 2011). Modify the properties of MSCS, including their ability to stimulate the growth of blood vessels, during aging and/or chronic pathologies may reduce the efficacy of autologous cell therapy in elderly patients with ischemic heart disease or chronic lower limb ischemia - n�the most likely candidates for cell therapy.

This necessitates the development of a method of pre-transplant evaluation of the angiogenic properties of its own progenitor cells of patients, in particular the MSC in adipose tissue, to determine the best course of cell therapy of ischemic diseases using these cells and select methods of modification of cells. The solution to this problem is the invention.

The present invention has no analogues (search the same or similar to the proposed solutions of patents was conducted in databases of WIPO, USPTO, Espacenet).

Disclosure of the invention

Because the ability of progenitor cells, including MSC in adipose tissue, stimulating the growth of blood vessels due largely products of their wide range of angiogenic factors (Rehman, 2004; Gimble, 2007; Kondo, 2009; Rubina, 2009; Madonna, 2010), in the way of a comprehensive evaluation of the angiogenic potential of MSC in adipose tissue of patients with coronary artery disease were included methods of assessing angiogenic activity of total products of secretion cells (Example 2) and the measurement of the mRNA level and secretion in the culture medium protein of the major angiogenic growth factors such as growth factor vascular endothelial (VEGF), placental growth factor (PlGF), a growth factor for hepatocytes (HGF), angiopoietin-1, angiogenin, for which various studies have shown Pro-angiogenic effect (P�emer 3). Since all of these factors act on the processes of angiogenesis, not individually, but in aggregate, another criterion for the evaluation of angiogenic potential of progenitor cells is the expression of angiogenic activity of total products of secretion, by which means the totality of substances, secreted by cells into the culture medium, composed of these Pro-angiogenic growth factors, and other excreted factors. To account, in addition to the production of paracrine factors, the contribution of intercellular interactions progenitor cells with other cells in their angiogenic potential in an integrated method of assessment was included to test the ability of MSC in adipose tissue to stimulate the growth of blood vessels in vivo in models of vascularization of subcutaneous implants of Matrigel entered immunodeficient mice (Example 4).

The object of the invention is to provide a more informative way to assess the angiogenic potential of progenitor cells in patients with cardiovascular disease for the prediction of therapeutic efficacy of cell therapy using these cells to treat ischemic diseases.

The technical result, which directed the claimed invention, is the possibility of obtaining more objective and detailed data on angiogenic sweat�ncial progenitor cells of the adult organism by obtaining various laboratory methods of quantitative results in the production of these cells the key factors stimulating angiogenesis, both at the level of gene expression and secretion in the culture medium, and use for assessment of functional tests stimulation of angiogenesis in the well-known science models in vitro and in vivo. Additional technical result is the possibility of simpler, faster and less costly, although less informative proximate analysis of the angiogenic potential of progenitor cells is disclosed in the invention, a method through the use of one of the functional tests included in the comprehensive assessment, the results of which significantly correlated with the results of other tests.

It was found that:

(a) angiogenic activity of total products of secretion of MSC in adipose tissue of patients with coronary artery disease decreases with age (Example 2);

b) the mRNA content of these Pro-angiogenic growth factors, PlGF and HGF, decline with age in MSC in adipose tissue of patients with coronary artery disease (Example 3);

b) the content of such Pro-angiogenic growth factors like VEGF, PlGF, HGF, angiopoietin-1 and angiogenin, decline with age in conditioned medium from MSC in adipose tissue of patients with coronary artery disease (Example 3);

g) the ability of MSC in adipose tissue to stimulate the vascularization of subcutaneous implants of Matrigel decreases with age of patients;

d) an indicator of angiogenic activity of total products of secretion of MSC in adipose tissue doscover� correlated with both the level of cells to produce Pro-angiogenic growth factors (Example 3), and with the ability of MSC in adipose tissue to stimulate the vascularization of subcutaneous implants of Matrigel (Example 4), which allows us to offer this method as an Express variant of the invention.

Use the Express method allows you to accelerate and reduce the cost of a method for evaluating the angiogenic potential of progenitor cells from a particular patient, and also to save a part of the cell material, which is used to implement the techniques included in the comprehensive evaluation of the angiogenic potential of these cells.

However, it was shown that to implement the ability of MSC in adipose tissue to stabilize the forming blood vessels is not enough only effects produced by them, angiogenic factors, it is necessary the formation of intercellular contacts between MSC in adipose tissue and other cells of the vascular wall (Rubina et al., 2009). Therefore, the method of rapid assessment of angiogenic potential of progenitor cells, reflecting the impact of paracrine factors, is more simple and accessible, but less accurate compared with the method of comprehensive evaluation.

A rapid method for evaluation of angiogenic properties of progenitor cells can be used as a screening to identify patients whose cells have a reduced angiogenic potential. In this case, the patient may be recommended a comprehensive method to value�key angiogenic potential of progenitor cells to identify targets for therapeutic correction of the properties of the cell material using the methods of modification of pre-transplant cells. As the latter can be used common methods of stimulation of regenerative, including angiogenic, properties of progenitor cells, such as cell culture at low oxygen content (1-5%) (Traktuev, 2006; Buravkova 2009; Rehman. 2004; Thangarajah, 2009; Efimenko, 2011), treatment of progenitor cells with statins, which activate Akt/eNOS intracellular signaling cascade (Dimmeler, 2001), or an inhibitor of protein kinase 38 (Seeger, 2005), which contributes to the proliferative activity of these cells, and culturing progenitor cells in the presence of a hormone of the pineal gland, melatonin, resulting in increased viability and proliferative activity of cells, increases their secretion of angiogenic factors and growth stimulation of blood vessels and astrocytes in the ischemic parenchyma of the kidney (Mias, 2008). Promising is the use of different methods of genetic modification of cells with constructs carrying the genes of angiogenic growth factors, the production of which is reduced in a given patient. In some cases, if the result of a comprehensive evaluation of the angiogenic potential of progenitor cells is very low, the patient may be recommended cell therapy using allogeneic cells from young healthy donors.

Conclusion about the specific level of the angiogenic potential of progenitor�'s cells in patients with cardiovascular diseases make at the stage of analysis of the results of all tests (in the case of a comprehensive assessment) or a single test (in the case of Express-analysis) (Implementation of the invention).

Brief description of figures

Fig. 1. Micrograph, reflecting the formation of tubular structures by endothelial cells on Matrigel in the presence of conditioned medium from MSC in adipose tissue of patients with coronary artery disease of different ages. As endothelial cells are used: A, B - endothelial cells of the human umbilical vein (HUVEC), G - endothelial cell line EA.hy926.

Fig. 2. Diagram and micrograph reflecting the results of the evaluation of the ability of MSC in adipose tissue of patients with coronary artery disease of different ages to stimulate the growth of blood vessels on the model of subcutaneous implantation of Matrigel immunodeficient mice. MSC in adipose tissue of CAD patients of different ages were mixed with Matrigel, depleted of growth factors in the amount of 7×105cells on the implant and injected subcutaneously to mice lines NUDE. MSK-Itmol - aunt patients younger than 60 years, MSC Jetstar - cells of patients older than 60 years. The implants were analyzed after 10 days. A - score is the number of CD31-positive formations on sections of the implants of Matrigel (n=16, 5 slices on the sample). The data are presented as median (10th, 90th percentiles). * - a 0.05<R<0,1. B - Immunohistochemical coloring frozen sections of implants of Matrigel containing MSC in adipose tissue of patients of different ages, on markers of endothelial cells (CD31, green fluorescence) and pericytes (NG2, red fluorescence), lens�Yves X40. The nuclei are stained DAPI (blue fluorescence).

The implementation of the invention

The implementation of the invention, in addition to the methods disclosed in detail in the following examples, were used well known in the art methods for culturing cells, also described in cited sources.

Example 1. Isolation and cultivation of MSC in adipose tissue of patients with CHD

MSC in adipose tissue was isolated from subcutaneous fat of patients (n=31) with coronary heart disease, stenosing coronary atherosclerosis on coronary angiography data. stable angina of II-IV functional class (according to the classification of the Canadian society for the study of cardiovascular disease) who underwent coronary artery bypass grafting. All patients with CHD were divided into 3 groups according to age: patients aged 44-48 years (subgroup And the average age 46,0±1,67 years old, n=6), 52-58 years (subgroup B, with an average age of 55.4±1.9 years, n=10) and older than 60 years (subgroup, the average age 68,9±4.2 years, n=15). According to the main clinical and anamnestic characteristics, namely gender, body mass index, functional class of angina, the frequency of myocardial infarction in anamnesis, presence of obesity, hypertension, diabetes type 2 diabetes and impaired glucose tolerance, dyslipidemia, subgroups of CHD patients of different ages did not significantly differ between with�battle.

Isolation of cells was performed according to previously published Protocol (Zuk et al., 2001) with some modifications. Subcutaneous fat obtained during coronary artery bypass grafting, were placed in a sterile tube. Isolation of cells was performed under sterile conditions in a laminar box. The fabric was shredded vascular scissors to the consistency of a suspension of small (no larger than a few cubic millimeters) pieces and mixed with solutions of the enzymes collagenase type I (200 u/ml, Worthington Biochemical, USA) and dispose (40 u/ml, Invitrogen Corporation, Germany) at a ratio of tissue volume (in ml) of enzyme solution (ml) 1:2. The sample is incubated at 37°C for 30 min under constant shaking; after incubation thereto was added an equal volume of medium AdvanceSTEM™ (HyClone) (#SH30879.01) and centrifuged at 200 g for 10 min. Whitish surface layer, the Mature adipocytes and slices of raw enzymatic tissue was removed, and the residue, consisting of stromal cells of adipose tissue, as well as the remnants of connective tissue and blood cells suspended in lysing buffer for red blood cells. The resulting mixture was incubated for 2-3 minutes at 37°C with stirring, after which the sample was added an equal volume of medium AdvanceSTEM™ (HyClone) (#SH30879.01) and was filtered through a nylon membrane (BD Falcon Cell Stainer) with a pore size of 100 m�crowns. For purification of a population of stromal cells from blood cells and cellular debris on the destination ethane suspension was centrifuged at 200 g for 5 min. Supernatant was removed, and the pellet was resuspended in the medium AdvanceSTEM™ (HyClone) (#SH30879.01) / Antibiotic / Antimycotic 100x Solution (HyClone) (#SV30079.01). Then transplanted the cells on Petri dishes (Corning) with a diameter of 60 mm in an amount of 1×105cells per dish and cultured in 4 ml medium AdvanceSTEM™ (HyClone) (#SH30879.01) / Antibiotic / Antimycotic 100x Solution (HyClone) (#SV30079.01) with the addition of 10% of the mixture of growth factors (Advance Stem Cell Growth Supplement, HyClone). The next day the culture medium was changed to fresh medium of the same composition and the cells were grown in CO2incubator (5% CO2; 95% air) at 37°C. the Change of medium was performed every 2-3 days; when reaching the monolayer cells were passively using HyQTase (HyClone) (#SV30030.01). Cells released from adipose tissue by processing enzymes, represent a mixed population of different cell types; however, when cultured in medium that supports growth of undifferentiated MSCS to the second passage in culture remain cells with fibroblast-like morphology, with noticeable differences in the morphology of cells derived from patients of different age groups, have been identified.

To obtain conditioned media, the cells of the second passage within 24 hours depreciable in the environment �OST, not containing serum, and then changed the environment to fresh and also not containing serum and cells were cultured for 48 hours. After that, the medium was collected, centrifuged at 200 g for 5 min, was added a cocktail of protease inhibitors (1:500, Sigma, cat. No. R), aliquots of 1.5 ml were frozen in liquid nitrogen and stored at -70°C.

Example 2. Evaluation of the angiogenic activity of total products of secretion of MSC in adipose tissue of patients with coronary artery disease

Angiogenic activity of total products of secretion of MSC in adipose tissue in the culture medium was assessed using the methods of education capillarity structures on Matrigel, depleted of growth factors (Growth factor reduced Matrigel, BD Biosciences) endothelial cells from the human umbilical vein (HUVEC) or the endothelial cell line EA.hy926 [Aranda, Owen, 2009]. For this pre-deprived for 18 hours endothelial cells of the umbilical vein (HUVEC) 2-4 passage or endothelial cell line EA.hy926 were seeded on 48-well plate, covered with Matrigel, in an amount of 2×104cells per well. Then to each well was added with conditioned medium from MSC in adipose tissue (in the case of HUVEC in a ratio of 1:1 with growth medium HUVEC). Each sample of conditioned medium was used in at least two holes. As a negative control, the environment of growth of endothelial cells that do not contain a serum as positive control - the HUVEC growth medium containing 20% FBS, or DMEM with low glucose with the addition of 50 ng/ml human VEGF. The tablet was placed in the CO2-incubator at 37°C for 24 h, and then analyzed the education capillarity structure using a light microscope (Leica, Germany). The total length of tubular structures in 5 randomly selected fields of view in each well were counted on the images obtained when using the X10 lens, using the program MetaMorph 5.0 (Universal Imaging).

It was found that the ability of conditioned media from MSC in adipose tissue of patients with coronary artery disease to stimulate the formation of tubular structures by endothelial cells on Matrigel in vitro (Fig. 1) decreases with age, which was reflected in the inverse correlation between the total length of the tubular structures and the age of patients (r=-0,53, p=0.03). Conditioned medium from cells isolated from adipose tissue of patients of older age (>60 years), stimulated the formation of tubular structures was significantly less pronounced than medium from cells obtained from younger patients (Table. 1).

Example 3. Measurement of the content of mRNA and proteins of the major angiogenic growth factors in MSC in adipose tissue of patients with coronary artery disease

The content of MSC in adipose tissue mRNA of factors involved in the regulation of angiogenesis. conducted by PCR in real time. For this�about from cells RNA was isolated using the RNeasy kit Miny Kit (50) (QIAGEN, USA), then on the matrix RNA cDNA built using the set of Fermentas Reverse Transcription Reagents (Fermentas, Lithuania). Next performed real-time PCR using intercalating dye SYBR Green I ("Synthol", Russia) and in the amplifier BIO-RAD iQ5 Multicolor Real-time PCR detection system (Bio-rad, USA). As primers used a unique complementary pairs of oligodeoxynucleotides to the analyzed genes (Table. 2). Data for each sample was normalized according to the expression of genes b-actin and gapdh.

We analyzed the mRNA content of genes are the main regulators of angiogenesis in MSC in adipose tissue of patients of different age groups. The results are presented in table. 3.

We observed a statistically significant decrease in the content of PlGF mRNA and the increase in the content of mRNA of PAI-1 in MSC in adipose tissue of patients of older age, while the content of mRNA of genes of other Pro - and antiangiogenic factors with age varied slightly. However, the content of VEGF mRNA (r=0.31, p=0.03) and PlGF (r=0,54, p=0.001) and HGF (r=0,39, p=0,09) correlated with angiogenic activity indicator total products of secretion of the corresponding MSC in adipose tissue. There was significant variation among cells of patients in their contents antiangiogenic factor endostatin (ENDS), however, further analysis showed that the air-conditioned rooms an�I Wednesday from MSC in adipose tissue with low levels of mRNA ENDS better stimulates the formation of tubular structures by endothelial cells (r=-0,46, p=0.05). This confirms data from the literature on the antiangiogenic activity of endostatin (O'reilly, 1997).

Most of the angiogenic growth factors are sekretiruemyi proteins, therefore, we evaluated the VEGF, PlGF, HGF, angiopoietin-1 (Angpt1), angiogenin content, thrombospondin-1 (TBS1) and plasminogen activator inhibitor-1 (PAI-1) in conditioned media from MSC in adipose tissue of patients of different ages (Table. 4).

The content of these factors was assessed using sets of reagents for enzyme-linked immunosorbent assay (ELISA) from R&D Systems (USA). To do this, conditioned medium obtained by culturing for 48 hours previously disadvantaged MSC in adipose tissue was defrosted. The measurement was performed as follows: 96-well plates containing antibodies against the studied protein was incubated with conditioned media samples or the standards of the studied factors as a positive control for 2 hours. After four times washing, special cleaning solution was applied biotinylated polyclonal antibody goat in a concentration of 0.2 μg/ml for two hours, and then repeated the procedure of the washing. As an enzyme that binds to biotinylating antibodies used streptavidin conjugated with horseradish peroxidase, diluted 1:200 with which the samples were incubated for 20 minutes For the development of the enzymatic reaction, the wells were added substrate for 20-30 min, then the reaction was terminated by addition of stop solution. The level of absorption of the solution in the wells was determined at a wavelength of 450 nm with correction at a wavelength of 620 nm. The results for different dilutions of standards relevant proteins were building a calibration curve, which determined the concentration of the studied protein in the conditioned media samples and counted by the number of cells for each sample. The results are presented in table. 4.

Despite the fact that the average values of the content of angiogenic factors in the environment of the cultivation of MSC in adipose tissue were several times lower in the older age subgroups than in the younger subgroups, significant differences for most factors due to the pronounced variation is not obtained. The exception is the level of angiogenin content that in patients older than 60 years was significantly reduced by more than three times compared with patients younger subgroup (42-48 years) (Tab. 4). However, correlation analysis showed the presence of accurate or close to accurate feedbacks between the content of Pro-angiogenic factors VEGF, PlGF, HGF, Angpt1 and angiogenin content in conditioned medium from MSC in adipose tissue and age of the patients with ischemic heart disease (Table. 4).

In order to assess how the content of angiogenic factors in conditioned medium�e related to its ability to stimulate the formation capillarity structures by endothelial cells, we compared for each sample of conditioned medium the concentrations of different growth factors and angiogenic activity of total products of secretion. It turned out that the strongest positive correlation was observed for angiopoietin-1 (r=0,79, p=0.004), but close to significant correlation was also observed between angiogenic activity of total products of secretion and content of angiogenin content (r=0.50, p=0,06), VEGF (r=0,65, p=0.04) and PlGF (r=0.56, p=0.08).

Example 4. Assessment of the ability of MSC in adipose tissue of patients with coronary artery disease to stimulate vascularization of subcutaneous implants of Matrigel entered immunodeficieny mice

Previously described several angiogenic mechanisms of action of MSC in adipose tissue, among which the production of angiogenic growth factors is the main but not the only mechanism that ensures the ability of MSC in adipose tissue to stimulate the growth of blood vessels. Therefore, to fully characterize the angiogenic properties of MSC in adipose tissue of patients with CHD, we used a model of angiogenesis in vivo (injection of cells consisting of subcutaneous implants of Matrigel immunodeficient mice).

For this 7×105MSC in adipose tissue of the second passage, obtained from patients younger than 60 years (MSK-Itmol) 60 years and older (GMT-Jetstar), were injected subcutaneously in immunocompromised mice lines NUDE (n=8) in the form of a suspension in Matrigel, depleted of growth factors (Growth really an reduced Matrigel, BD Biosciences), using an insulin syringe with needle thickness 23G so that Matrigel was time to polymerize under the skin and form a gelatinous implant irregular shape (2 of the implant on the animal).

10 days after subcutaneous injection of cell suspension in Matrigel mice were killed by cervical dislocation, fully implants removed and frozen in liquid nitrogen in the medium for frozen tissue Tissue-Tek (Sakura, Japan) for subsequent preparation of sections (6 µm). Assessment of the density of blood vessels in sections of Matrigel was performed using immunofluorescent staining using monoclonal rat antibodies against marker antigen vascular endothelial CD31 (BD Pharmigen, cat. No. 550274, USA) and rabbit antibodies against the marker of pericytes NG2 mouse (Chemicon, cat. No. ab5320, USA). The nuclei are counterstained with a fluorescent dye DAPI. The obtained preparations were analyzed using a fluorescence microscope ZeissAxiovert200M. Documentation of images produced with a digital camera Axiocam HRc (Zeiss, Germany) and processing in the Axiovision program. The size of the vessels and their density was estimated using the software MetaMorph 5.0 (Universal Imaging) and ClickCounter. The counting of blood vessels was performed in 5 fields of view on the slice (12 slices for each implant) at 20 × magnification, separately highlighting the capillaries (CD31-put� - to-date education without a gap or length less than 20 microns), the medium diameter vessels (CD31-positive education with a diameter or a length of 20-50 microns) and large vessels (diameter greater than 50 microns). The number of vessels was normalized per unit area of the slice.

Using double immunofluorescent staining of sections of Matrigel antibodies against markers of vascular endothelial CD31 and pericytes NG2 was found to MSC in adipose tissue of older patients, on average, one and a half times weaker stimulated vascularization of implants than MSC in adipose tissue of younger patients (Fig. 2), although the differences did not reach statistical significance (mean density of all vessels 47,7 (41,2; 49,9) vs. 69,0 (60,0; 73,6), respectively, p=0,09; the average density of capillaries and 36.2 (31,9; 36,2) vs. 59,0 (54,3; and 62.6, respectively, p=0,06) (Fig. 2).

We have identified the differences between angiogenic activity of MSC in adipose tissue obtained from patients with coronary artery disease of different ages, confirm the need for a preliminary assessment of cellular material for autologous cell therapy of ischemic diseases.

It should be noted that when comparing the results of the evaluation of the angiogenic activity of total products of secretion of MSC in adipose tissue of patients and the ability of the respective sample cells to stimulate vascularization of subcutaneous implants of Matrigel (total number of vessels on the analyzed sections of the implant) we found that these results�whether significantly intercorrelated (r=0,54, p=0.03, n=16). Given that the angiogenic activity of total products of secretion was also significantly correlated with the level of the main Pro-angiogenic growth factors produced by MSC in adipose tissue, it is proposed to use as a rapid assessment of the angiogenic properties of MSC in adipose tissue method for the formation of tubular structures by endothelial cells in the presence of conditioned medium from MSC in adipose tissue.

Thus, the method of complex evaluation of the angiogenic potential of progenitor cells in patients with cardiovascular disease according to the present invention includes (a) determining the content of mRNA and proteins of the most important Pro-angiogenic growth factors (VEGF, PlGF, HGF, angiopoietin-1, angiogenin content); b) measuring angiogenic activity of total products of secretion cells; b) assessing the ability of cells to stimulate vascularization of subcutaneous implants of Matrigel entered immunodeficient mice. Screening can be used more simple and affordable method for the rapid assessment of angiogenic properties of MSC in adipose tissue by measuring angiogenic activity of total products of secretion cells, given the fact that this index was significantly correlated with both the level produced by MSC in adipose tissue Pro-angiogenic growth factors and the ability of MSC in adipose tissue to promote tissue vascularization in vivo. However, it is not from�Ajeet differences in the ability of MSC in adipose tissue to stabilize the emerging blood vessels and is less accurate compared with the method of comprehensive evaluation.

Stage, the assessment of angiogenic potential of progenitor cells is the analysis of quantitative data obtained as a result of all these tests (in the case of a comprehensive assessment) or a single test (in the case of Express-analysis). It is known that the average age of CHD onset is 45-50 years (American Heart Association, 2002), therefore, indicators of patients with ischemic heart disease age group of 42 to 48 years (mean age 46,0±1,67 years) are considered as normal. Thus, given the results of these studies are presented in Table III. 1, the results of proximate analysis are normal for patients with cardiovascular disease when the measured values of index ≥89143 Rel.ed and reduced when the values of the measured indicator <89143 Rel.ed. (see Table 1).

In the case of a comprehensive assessment of the final results of the measurements are considered for patients with cardiovascular diseases normal under the following measurement values: VEGF ≥2.4 Rel.ed. (mRNA) and ≥23 ng/million cells (secretion), PlGF ≥6,6 Rel.ed. (mRNA) and ≥0.5 ng/million cells (secretion), HGF ≥2.5 Rel.ed. (mRNA) and ≥20 ng/million cells (secretion), angiopoietin-1 ≥2.4 Rel.ed. (mRNA) and ≥1.0 ng/million cells (secretion), angiogenin ≥0,4 Rel.ed. (mRNA) and ≥6.8 ng/million cells (secretion), angiogenic activity of total product�tov secretion ≥89143 Rel.ed., the level of vascularization of the implants of Matrigel by the total number of vessels ≥69 Rel.ed. (see Tables 1, 3, 4 and the text description on page 13).

The final results of the measurements are considered for patients with cardiovascular diseases reduced under the following measurement values: VEGF <a 2.4 Rel.ed. (mRNA) and <23 ng/million cells (secretion), PlGF <6,6 Rel.ed. (mRNA) and <0.5 ng/million cells (secretion), HGF <2.5 Rel.ed. (mRNA) and <20 ng/million cells (secretion), angiopoietin-1 <2.4 Rel.ed. (mRNA) and <1.0 ng/million cells (secretion), angiogenin <0,4 Rel.ed. (mRNA) and <6,8 ng/million cells (secretion), angiogenic activity of total products of secretion <89143 Rel.ed., the level of vascularization of the implants of Matrigel by the total number of vessels <69 Rel.ed (see Tables 1, 3, 4 and the text description on page 13).

The proposed method can be further used for testing autologous cell material obtained from patients, including coronary heart disease, prior to transplantation to choose the best course of cell therapy to stimulate the growth of blood vessels.

The list of references

1. Buravkova L. B., Grinakovskaya O. S., Andreeva, E. R. et al. Characterization of mesenchymal stromal cells from human lipoaspirate, cultured under reduced oxygen content. �etologia. 2009; 51(1):5-10.

2. Efimenko A., Starostin, E. E., Kalinina N. And., Parfenova E. V. the Influence of age on angiogenic properties of mesenchymal stem cells from adipose tissue. Cell transplantation and tissue engineering. 2011; VI (3):48-57.

3. Efimenko A., Starostin, E. E., Rubin, C. A., Kalinina N. And., Parfenova E. V. Influence of hypoxia and inflammatory factors on the viability and angiogenic activity of mesenchymal stromal cells from adipose tissue and bone marrow. Cytology. 2010; 52(2):144-154.

4. Kalinina N. And., Efimenko A., Starostin, E. E., Parfenova E. V., Tkachuk V. A. Hypoxia as the main activator of angiogenesis and growth of adipose tissue. Russian physiological journal. I. M. Sechenov. 2009; 95(3):283-9.

5. Parfenova E. V., Tsokolaeva Z. I., Traktuev D. O., etc., the Search for new "tools" for therapeutic angiogenesis. Molecular medicine. 2006; 2:10-23.

6. Ruby K. A., Kalinina N. And., Efimenko A., Lopatina T. V., Melikhov V. A.. Tsokolaeva Z. I., Sysoev, V., Tkachuk V. A., Parfenova E. V. the Mechanism of stimulation of angiogenesis through the ischemic myocardium by stromal cells of adipose tissue. Cardiology. 2010; 50(2):51-61.

7. Traktuev D. O., Parfenov, E. V., Tkachuk V. A. et al. Stromal cells of adipose tissue - plastic type of cells with high therapeutic potential. Cytology. 2006; 48:83-94.

8. Aranda, E., Owen G. I. A semi-quantitative assay to screen for angiogeni compounds and compounds with angiogenic potential using the EA.hy926 endothelial cell line. Biol Res. 2009; 42:377-389.

9. Bailey A. M., Kapur, S., Katz A. J. Characterization of adipose-derived stem cells: an update. Curr Stem Cell Res Ther. 2010; 5(2):95-102.

10. Cai L., Johnstone, B. H., T. G. Cook, et al. IFATS collection: Human adipose tissue-derived stem cells induce angiogenesis and nerve sprouting following myocardial infarction, in conjunction with potent preservation of cardiac function. Stem Cells. 2009; 27(1):230-7.

11. Dimmeler S., Aicher A., Vasa, M. et al. HMG-CoA reductase inhibitors (statins) increase endothelial progenitor cells via the PI 3-kinase / Akt pathway. J Clin Invest. 2001; 108(3):391-7.

12. Efimenko A. Yu., Starostina E. E., Rubina K. A., Kalinina N. I., Parfenova E. V. Viability and Angiogenic Activity of Mesenchymal Stromal Cells from Adipose Tissue and Bone Marrow under Hypoxia and Inflammation in vitro. Cell and Tissue Biology. 2010; 4; 2:117-127.

13. Efimenko A. Starostina E., Kalinina N, Stolzing A. Angiogenic properties of aged adipose derived mesenchymal stem cells after hypoxic conditioning. Journal of Translational Medicine. 2011; 9(1):10-22.

14. El-Ftesi S., Chang E. I., Longaker M. T., et al. Aging and diabetes impair the neovascular potential of adipose-derived stromal cells. Plast Reconstr Surg. 2009; 123(2):475-85.

15. Gimble J. M., Bunnell, B. A., E. S. Chiu, F. Guilak Concise Review: Adipose derived stromal vascular fraction cells and stem cells: let's not get lost in translation. Stem Cells. 2011; 29(5):749-54.

16. Gimble J. M., Katz A. J., Bunnell B. A. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007; 100(9):1249-60.

17. Heart Disease and Stroke Statistics-2003 Update. - Dallas: American Heart Association, 2002.

18. Huang S. C., Wu T. C., Yu H. C., et al. Mechanical strain modulates age-related changes in the proliferation and differentiation of mouse adipose-derived stromal cells. BMC Cell Biol. 2010; 11 to 18.

19. Kachgal S., Putnam, A. J. Mesenchymal stem cells from adipose and bone marrow promote angiogenesis via distinct cytokine and protease expression mechanisms. Angiogenesis. 2011; 14(1):47-59.

20. Katsara, O., Mahaira L. G., Iliopoulou E. G., et al. Effects of Donor Age, Gender, and In Vitro Cellular Aging on the Phenotypic, Functional, and Molecular Characteristics of Mouse Bone Marrow-Derived Mesenchyml Stem Cells. Stem Cells Dev. 2011; in press.

21. Kondo K., Shintani S., Shibata, R., et al. Implantation of adipose-derived regenerative cells enhances ischemia-induced angiogenesis. Arterioscler Thromb Vasc Score Biol. 2009; 29(1):61-6.

22. Madonna R., De Caterina R. Adipose tissue: a new source for cardiovascular repair. J Cardiovasc Med (Hagerstown). 2010; 11(2):71-80.

23. Madonna R., Y. J. Geng, De Caterina R. Adipose tissue-derived stem cells: characterisation and potencial for cardiovascular repair. Arterioscler Thromb Vasc Score Biol. 2009; 29:1723-1729.

24. Madonna R., F. Renna, C. Cellini, et al. Age-dependent impairment of number and angiogenic potential of adipose tissue-derived progenitor cells. Eur J Clin Invest. 2011; 41(2):126-33.

25. Mias S., Trouche E., Seguelas M. H. et al. Ex vivo pretreatment with melatonin improves survival, proangiogenic/mitogenic activity, and efficiency of mesenchymal stem cells injected into ischemic kidney. Stem Cells. 2008; 26(7):1749-57.

26. Miranville A., Heeschen C., Sengenes C., et al. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells. Circulation. 2004; 110(3):349-55.

27. Miyahara Y., Nagaya N., Kataoka, M., et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med. 2006; 12(4):459-65.

28. Murohara, T., Shintani S., Kondo K. Autologous adipose-derived regenerative cells for therapeutic angiogenesis. Curr Pharm Des. 2009; 15(24):2784-90.

29. Nakagami h, Morishita R., Maeda, K., et al. Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy. J Atheroscler Thromb. 2006; 13(2):77-81.

30. O'reilly M. S., Boehm T, Shing y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997, 88(2):277-285.

31. Planat-Benard V, Silvestre, J. S., Cousin V., et al. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 2004; 109(5):656-63.

32. Rehman j, Traktuev d, Li J., et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation. 2004; 109(10):1292-8.

33. Rubina K. A., Kalinina N. I., Efimenko A. Y., Lopatina T. V., V. Melikhova A., Tsokolaeva Z. N., Sysoeva Y. V., Tkachuk V. A., Parfyonova Ye.V. AdiposeStromal Cells Stimulate Angiogenesis via Promoting Progenitor Cell Differentiation, Secretion of Angiogenic Factors, and Enhancing Vessel Maturation. Tissue Eng Part A. 2009; 15(8):2039-50.

34. Seeger, F. H., Haendeler J., Walter, D. H. et al. p38 mitogen-activated protein kinase downregulates endothelial progenitor cells. Circulation. 2005; 111(9):1184-91.

35. Sethe s, Scutt A., Stolzing A. Aging of mesenchymal stem cells. Ageing Res Rev. 2006; 5(1):91-116.

36. Stolzing, A., E. Jones, D. McGonagle, et al. Age-related changes in human bone marrow-derived mesenchymal stem cells: consequences for cell therapies. Mech Ageing Dev. 2008; 129(3):163-73.

37. Sumi M., Sata, M., Toya N., et al. Transplantation of adipose stromal cells, but not mature adipocytes, augments ischemia-induced angiogenesis. Life Sci. 2007; 80(6):559-65.

38. Sun Y., Li W., Lu Z., et al. Rescuing replication and osteogenesis imperfecta of aged mesenchymal stem cells by exposure to a young increasing interest among matrix. FASEB J. 2011; 25(5):1474-85.

39. Thangarajah, H., Vial, I. N., E. Chang, et al. IFATS collection: Adipose stromal cells adopt a proangiogenic phenotype under the influence of hypoxia. Stem Cells. 2009; 27(1):266-74.

40. Zhu M., Kohan, E., Bradley J., et al. The effect of age on osteogenic, adipogenic and proliferative potential of female adipose-derived stem cells. J Tissue Eng Regen Med. 2009; 3(4):290-301.

41. Zuk P. A., Zhu, M., Mizuno H., et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001; 7(2):211-28.

1. Method of complex evaluation of the angiogenic potential of progenitor cells in patients with cardiovascular diseases, including quantitative measurement of mRNA content and protein key Pro-angiogenic factors, growth factor vascular endothelial (VEGF), placental growth factor (PlGF), growth factor hepatocyte (HGF), angiopoietin-1 and angiogenin content produced by cells, angiogenic activity of total products of secretion cells containing at least the specified f�story, on the model of the formation of tubular structures by endothelial cells in the presence of conditioned media from progenitor cells, as well as evaluating their own abilities progenitor cells of the patient to stimulate the vascularization of subcutaneous implants of Matrigel entered immunodeficient mice, thus the conclusion about reduced angiogenic potential of doing with the following values of the parameters under study: VEGF <a 2.4 Rel.ed. (mRNA) and <23 ng/million cells (secretion), PlGF <6,6 Rel.ed. (mRNA) and <0.5 ng/million cells (secretion), HGF <2.5 Rel.ed. (mRNA) and <20 ng/million cells (secretion), angiopoietin-1 <2.4 Rel.ed. (mRNA) and <1.0 ng/million cells (secretion), angiogenin <0,4 Rel.ed. (mRNA) and <6,8 ng/million cells (secretion), angiogenic activity of total products of secretion <89143 Rel.ed., the level of vascularization of the implants of Matrigel by the total number of vessels <69 Rel.ed.

2. Method of Express-evaluation of the angiogenic potential of progenitor cells in patients with cardiovascular disease by measuring angiogenic activity of total products of secretion cells containing the key Pro-angiogenic factors comprising at least a growth factor vascular endothelial (VEGF), placental growth factor (PlGF), a growth factor for hepatocytes (HGF), angiopoietin-1 and angiogenin, on the model fo�of tubular structures by endothelial cells from the human umbilical vein or endothelial human cell line EA.hy926 in the presence of conditioned media from progenitor cells, the conclusion about reduced angiogenic potential of doing when the value of the investigated parameter below 89143 Rel.ed.



 

Same patents:

FIELD: medicine.

SUBSTANCE: group of inventions relates to field of biotechnology. Method of detecting genome of virus of infectious anaemia in horses includes carrying out polymerase chain reaction, amplification of virus DNA, evaluation of reaction carrying out. If viral RNA is detected, reaction of denaturation and reverse transcription is preliminarily carried out, and detection is carried out by means of RT-PCR in real time in case of detection of INAN virus and PCR in real time in case of detection of pDAN of INAN virus. To realise the method specific primers and DNA-probe are applied. Temperature modes include the following stages: 5 min of preliminary denaturation at 94°C, 5 cycles of amplification without detection (94°C - 10 sec, 56°C - 15 sec, 72°C - 15 sec) and 35 cycles of amplification with detection (94°C - 10 sec, 56°C - 15 sec, 72°C - 15 sec). Interpretation of results is carried out on the basis of presence/absence of crossing of fluorescence curve with threshold line (Threshold).

EFFECT: application of inventions makes it possible to detect RNA and pDAN of INAN virus by means of RT-PCR in real time mode, as well as to increase method specificity and sensitivity.

2 cl, 3 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: method is based on single nucleotide polymorphism of genes of toxin-anatoxin systems of type II of superfamilies VapBC, HigAB and MazEF and is characterised in that for identification amplification is carried out with genomic DNA using the set of oligonucleotide primers for 10 genes of toxin-anatoxin systems. PCR products are analysed in agarose gel, the size of the obtained fragment is determined by DNA marker, the target fragments are isolated from the PCR mixture or the agarose gel and they are sequenced in order to detect the respective polymorphisms.

EFFECT: invention enables to carry out fast and accurately genotyping of mycobacteria in PCR mode and can be used for identification of clinically important strains, including with multiple and wide drug resistance, and also with altered virulence in operation with patients.

1 dwg, 3 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to multi-channel devices, modified by nanocarriers of aniline-containing polymers. Claimed are: a multi-channel nozzle for the extraction of nucleic acids, proteins, peptides and a method of production of a multi-channel element, included in the composition of the multi-channel nozzle. The nozzle consists of a glass hexagonal multi-channel array, which includes multi-channel elements from a multitude of parallel capillaries. A sorption layer, which consists of one to three polymeric nanolayers, is applied on the internal surface of the capillaries. A multi-channel element is made with a protective framing from several rows of glass rods. The glass hexagonal multi-canal array consists of the laid repeatedly extended single multi-channel elements with the diameter of capillaries from 1 mcm and more, working surface to 500 cm2, volume to 500 mcl. The method of the multi-channel element production includes the assembly of glass tubes into a multi-channel pack, fastening one of its ends in a supply unit grip, heating of the other end in a furnace with the formation of a bulb and extension to a required size. In order to obtain the structures with the diameter of the channel of the order and less than 1 mcm in the process of moulding the repeated extension of the multi-channel elements, preliminarily formed by laying the elements of the hexagonal shape, is realised.

EFFECT: inventions ensure carrying out the express-extraction and purification of both nucleic acids and proteins, peptides, as well as ensure the high reproducibility of results.

5 cl, 6 dwg, 3 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology, virology and immunology. Particularly, the present invention refers to a new avian astrovirus; to antibodies and their fragments targeted to the above new virus; to antigen preparations, proteins and DNA molecules of new avian astrovirus; to vaccines based on the above new virus or to its antigen preparations, protein or DNA; to methods for producing such vaccines and to diagnostic kits. The present invention can be used in veterinary science.

EFFECT: preparing the new avian astrovirus.

11 cl, 5 dwg, 4 tbl, 12 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions relates to biotechnology. Claimed are: method of obtaining personified autoprobiotic product in form of lactic ferment based on autostrains of lactobacteria and method of treating syndrome of irritable bowl, accompanied by bowl dysbiosis, with its application. Claimed product is obtained by sampling native material, enoculation and growing of bacteria from patient's excrements on selective culture medium, identification and collection of typical for lactobacteria colonies, obtaining clear culture, specification of belonging to species by application of PCR, its deposition in cryostorage at temperature not higher than -75°C with storage term not more than 1 year and preparation lactic ferment from obtained culture. As lactobacteria applied are autostrains of Lactobacillus spp. For PCR specific for determined species of lactobacilla primers are applied. Lactic ferment if prepared from clear culture of obtained strains with titre not less than 1×108 CFU/ml. Obtained product is prescribed to subject in dose not less than 50 ml with number of intakes not fewer than 2 times per day after 30-40 min after meal for not fewer than 10 days.

EFFECT: group of inventions makes it possible to increase duration of stable treatment and its efficiency.

2 cl, 5 tbl

FIELD: medicine.

SUBSTANCE: presented group of inventions refers to medicine. What is presented is a method for the prediction of a high probability of the therapeutic effect of high-affinity anti-VEGF antibody, particularly ranibizumab in the patient suffering age-related moist macular degeneration. What is presented is a kit comprising a first oligonucleotide and additional nucleotides specific for allele polymorphisms of matrix metalloprotease 25 (MMP25) gene corresponding to rs1064875. If the respective genotype contains AA or AG, the high probability of the effect of the above treatment is predicted.

EFFECT: presented group of inventions provides the effective agents and methods for the prediction of the high probability of the effect of the treatment with the high-affinity anti-VEGF antibody in the patient with moist AMD.

9 cl, 1 dwg, 5 tbl, 1 ex

FIELD: biotechnology.

SUBSTANCE: invention is the mutant variants of recombinant L-asparaginase characterised by amino acid sequence corresponding to the amino acid sequences of L-asparaginase of bacteria Wolinella succinogenes, in which the lysine amino acid residue in the position 24 is replaced by the serine residue or the valine amino acid residue in the position 23 is replaced by the glutamine residue, and the lysine amino acid residue in the position 24 is replaced by the threonine residue.

EFFECT: invention enables to obtain variants of L-asparaginase with improved resistance to proteolysis under the action of trypsin when compared to the unmodified recombinant protein and different levels of glutaminase activity while complete maintaining asparaginase activity, which makes them attractive objects for development on their basis of new antitumor therapeutics preparations.

2 cl, 1 dwg, 1 tbl, 11 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a set of oligonucleotide primers for amplification of a section of genome of pathogenic burkholderia, the sequence of which is homological to a fragment of genome B. mallei BMAA1526, for detection by means of sequenation of the change of the number of tandem arrays of DNA of strains of maliasmus and melioidosis exciters.

EFFECT: invention allows detecting changes in a genome of strains of maliasmus and melioidosis exciters, which are cultivated under different conditions on nutritive media and subcultured on laboratory animals.

2 dwg, 2 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a set of oligonucleotide primers for amplification of a section of genome of pathogenic burkholderia, the sequence of which is homological to a fragment of genome B. mallei BMAA0090, for detection by means of sequenation of the change of the number of tandem arrays of DNA of strains of maliasmus and melioidosis exciters.

EFFECT: invention allows detecting changes in a genome of strains of maliasmus and melioidosis exciters, which are cultivated under different conditions on nutritive media and subcultured on laboratory animals.

2 dwg, 2 ex

FIELD: process engineering.

SUBSTANCE: set of invention relates to biology, particularly, to automated purification of biological specimens at extraction of target matters involving the magnetic field application. Magnetic field application element and heater are integrated for up and down movement. Automatic device comprises unit 100 displacing vertically and horizontally to accommodate pipettes 141, 142 for suction and discharge of fluid. Heater 810 heats a particular unit of holes of multihole tray 420, 420' while element 700 for magnetic field application is arranged on support plate 400 including magnet seat 710. Element 700 accommodates magnet 711 at bottom side of said unit of said tray Proposed method exploits above described device.

EFFECT: higher process efficiency.

26 cl, 48 dwg

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology. A method provides preparing a suspension of pluripotent stem cells and adding a compound able to inhibit Rho-kinase activity to the suspension. That is followed by adding the cell suspension to a flat carrier, and the compound is removed after the cells has been fixed.

EFFECT: what is described is the method for human pluripotent stem cell fixation on the flat carrier.

14 cl, 32 dwg, 10 tbl, 14 ex

FIELD: biotechnology.

SUBSTANCE: method of detection of intracellular proteins using fluorescein-5-isothiocyanate (FITC) in various types of mammalian cells is provided, which are characterised by different levels of the synthetic process, using the method of confocal laser microscopy. The mammalian cells are fixed by paraformaldehyde (PFA) preventing the extraction of proteins in subsequent stages of staining, permeabilised with detergent, then stained for 2 h with FITC in a concentration of 1 mcg/ml. The cells are enclosed in Mowiol 4-88 adding DABCO (1,4-diazabicyclo[2.2.2] octane). The resulting preparations are used to analyse the localisation and the content of the protein component of the cells by confocal microscopy.

EFFECT: invention enables to obtain information and to investigate the location of proteins in the cells and to carry out a comparative analysis of protein content in cells with different levels of metabolism.

7 dwg, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to compositions for intensive generation of a target protein in eucariotic cells, which includes a DNA vector with an insert of target protein gene and an agonist of cell receptors. Besides, the invention relates to methods for increasing generation of a target protein coded with a transgene in eucariotic cells by using the above compositions.

EFFECT: invention allows effective increase of generation of a target protein in eucariotic cells.

28 cl, 4 dwg, 7 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, namely to orthopaedics, and can be used for producing a transplant for long bone tissue repair. That is ensured by collecting bone marrow aspirate into lithium heparin test tubes, diluting with phosphate salt buffer, filtered through a filter with pore size 70 mcm and centrifuging for 10 min at 400 g. That is followed by inoculating flasks 150 cm2 with the produced mesenchymal stem cells (MSCs) at 1×106 cells/cm2, culturing at temperature 37°C and 5% CO2 in air, in the Dulbecco's Modified Eagle's medium containing glucose 1 g/l and less, with 10% embryo calve serum. The medium is changed every 3 days. Before preparing the transplant, the cells are removed from the substrate with 0.05% trypsin EDTA, washed and re-suspended. The cell suspension is applied on the matrix, incubated for 3 hours at temperature 37°C with rocking in a rotation shaker at 25 rpm. The matrix is filled with the DMEM medium with 10% bovine foetal serum, dexamethasone 10-7 M, β-glycerophosphate 10 mM, ascorbate-2-phosphate 0.05 mM, 1% streptomycin 100 mcg/ml or penicillin 100 units/ml. The MSCs are cultured for 28 days. What is also presented is a method for long bone tissue repair.

EFFECT: group of inventions provides the uniform bone tissue repair in replacing the bone tissue defect of critical size with the transplant.

3 cl, 2 tbl, 6 ex

FIELD: veterinary medicine.

SUBSTANCE: presented subline of cells A4C2/9k is highly sensitive to the ASF virus. The growth medium is used as medium Needle-MEM with 10% blood serum of swine. The inoculating concentration is 80-100 thousand cells/ml. The mitotic index to 2-3 days of cultivation is 25-35. The subline of cells is deposited at the Specialized collection of cell cultures of agricultural and game animals at the All-Russian research institute of experimental veterinary medicine under the number of 87.

EFFECT: invention enables to isolate the African swine fever virus without prior adaptation in reaction of hemadsorption and provides its accumulation in titre.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to methods for cell and tissue protection against a hypoxic injury and can be used for developing remedies for hypoxic and ischemic injuries. The developed method for protection is based on cell treatment by substances increasing a level of glutathionylation of Na,K-adenosine triphosphatase catalytic subunit that reduces to its activation. The obtained results can be actual for biological research institutions, medical facilities, particularly cardiologic clinics, as well as biotechnological companies for transplantology and tissue engineering.

EFFECT: presented method can be used for research activities aiming at creating the therapeutic agents for relieving tissue injuries, particularly myocardial tissue in hypoxia and hypoxia/reoxygenation.

6 dwg

FIELD: medicine.

SUBSTANCE: invention refers to a method for developing Alzheimer disease cell models for testing medical effectiveness of chemical substances to be further used in medicine, more specifically in treating individual's neurodegenerative diseases caused by neurotoxic effects of beta-amyloid aggregates. A molecular cytotoxic agent is a synthetic analogue of human beta-amyloid 1-42 isomerised in asparagic acid residue in position 7 ([isoD]) (amino acid sequence: DAEFRH[isoD]SGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA). The additional advantage of the present invention is applicability of primary cell cultures as Alzheimer disease models.

EFFECT: advantage of these Alzheimer disease cell models as compared to currently used exogenous-induced models, wherein the cytotoxic effects are caused by a non-modified form of beta-amyloid is a higher ability of the isomerised beta-amyloid to induce both cell necrosis and apoptosis.

2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and concerns a method for producing progenitor cells from mammalian myocardial samples. A presented method involves milling the samples, implanting them on culture dishes coated so that to provide adhesion; culturing the samples in the hypoxic environment so that to provide the sample adhesion to form a cell culture; recovering cells from the cell culture by enzymatic treatment; culturing the recovered cells until forming cell aggregates, and processing the cell aggregates with an enzyme solution to produce the progenitor cells of myocardium.

EFFECT: presented invention can be used in medicine and veterinary science for studying the myocardium regeneration mechanisms and stem cell biology, as well as for treating cardiac diseases.

10 cl, 2 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and medicine. What is presented is the mus musculus's hybrid culture cell strain α-producing monoclonal antibodies specific to human granulocyte colony-stimulating factor (GCSF) deposited in Special Cell Culture Collection of Institute of Cytology of the Russian Academy of Sciences, under No. PKKK ("П") 662 "Д".

EFFECT: invention enables extending the range of strains producing GCSF-specific monoclonal antibodies used for research and medical applications.

2 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to gastroenterology, and can be applied as a method of treating hormone-dependent/hormone-resistant ulcerative colitis complicated by opportunistic infections. That is ensured by measuring herpes simplex virus type 1 IgG, herpes virus type 6 IgG, cytomegalovirus IgG, mycoplasm IgG, Chlamydia IgG. If herpes simplex virus type 1 IgG is 185 units/ml and more, and IgM is 0.4 units/ml and more, herpes virus type 6 IgG is 1.5 units/ml and more, cytomegalovirus IgG k is 185 units/ml and more, IgM is 3.4 units/ml and more, mycoplasm IgG is 119 units/ml and more, IgM is 0.4 units/ml and more, Chlamydia IgG is 105 units/ml and more, IgM is 0.8 units/ml and more, a systemic transplantation of allogenic mesenchymal stem cells in a dose of 150-250 million cells.

EFFECT: using the given method enables providing the more effective therapeutic treatment of hormone-dependent/hormone-resistant ulcerative colitis, promotes CMV elimination with no anti-viral therapy required and overcoming of hormone-dependency/hormone-resistance.

3 ex

FIELD: medicine.

SUBSTANCE: skin graft is simulated in laboratory animals on the second experimental day. Dihydroquercetin is administered intragastrically in a daily dose of 5.5 mg/kg from the first day every 46 hours of the experiment.

EFFECT: increasing the skin graft survival in the reduced circulation environment by activating the pre-conditioning process.

1 ex, 1 tbl

Up!