Method for locally suppressing angiogenesis in target tissue

FIELD: medicine; biology.

SUBSTANCE: method involves introducing vector containing DNA-sequence into area under protection encoding T-cadgerin and being capable of providing its expression in target tissue or cells compatible with target tissue cells transected with said vector in advance and selected with respect to T-cadgerin expression level. The method has been tested on model system by implanting Matrigel to mice. Reliable implant mass-, implant hemoglobin contents- and capillary and medium-sized blood vessel number reduction has been observed in experimental animals, receiving cells L929 (clone TC3) expressing T-cadgerin, in two weeks after the injection.

EFFECT: enhanced effectiveness in treating pathological states with new blood vessel formation being suppressed.

7 cl, 8 dwg

 

The present invention relates to the field of biotechnology, in particular genetic and cell engineering, and can be used in experimental biology and medicine. It is suggested the way the local suppression of angiogenesis, bringing the target tissue an effective amount of functionally active T-cadherin due to the overexpression of this gene encoding its gene introduced into the protected area (the target tissue).

Angiogenesis, the formation of new blood vessels in the branching and growth of preexisting blood vessels, accompanied by the normal formation of organs and tissues of human embryogenesis. This process can be divided into several stages: the migration and proliferation of endothelial cells, formation of new capillary sprouts, migration and proliferation subendothelial cells (pericytes), which causes the formation of a well-functioning, Mature vasculature. All these stages are controlled by a number of factors that stimulate or inhibit angiogenesis. These factors include soluble biologically active substances (cytokines and growth factors), as well as transmembrane proteins expressed on the cell surface (integrins and aminy) (Brantley-Sieders DM, and J. Chen, 2004). The imbalance positively the x and negative modulators of angiogenesis in tissues is one of the causes of such diseases, as psoriasis (T. T. Leong, 2005), retinopathy (Fischer-Colbrie R. et al., 2005), rheumatoid arthritis (Pap T. and O. Distler, 2005) and atherosclerosis (Collett GD and Canfield AE., 2005). Conversely, the introduction of externally stimulating or inhibiting angiogenesis factors can be directed to influence this process and is used to treat a number of diseases. Thus, the introduction of growth factors that stimulate angiogenesis, is used to restore blood supply to ischemic tissues, and the introduction of agents, inhibiting the growth of blood vessels, in the treatment of cancer because the tumor growth depends on the intensity of their vascularization.

The level of technology

There are various natural and synthetic compounds with the ability to suppress the formation of new blood vessels. Their action is usually directed at the inhibition of angiogenic growth factors (antibodies against VEGF, soluble forms of the receptors for VEGF and FGF), or to suppress the sensitivity of endothelial cells to growth factors (suramin, pentosan), or to block the holding of intracellular signals or inhibiting proliferation and migration of endothelial cells (Kiselev et al., 2003). However, the range of these compounds is currently quite limited. In addition, most known drugs used to suppress angiogenesis, has the polypeptide is riroda, making it difficult to obtain a stable directional effects in target tissues.

The problem of stabilization and containment of the effect of the polypeptide inhibitors of angiogenesis in recent years, partly resolved through the development of methods based on the introduction into the region of the target coding genes or containing these genes vector designs. In particular, for the suppression of tumor angiogenesis using the introduction replication-deficient viruses (adeno - and adeno-associated) and plasmids containing DNA that encode proteins inhibitors, for example, a fragment of collagen 18 endostatin, a fragment of plasminogen angiostatin and others (Seth P., 2005, Ponnazhagan, S., 2004). Any of these methods can be considered as an analogue of the present invention.

Methods of this group, however, have disadvantages that limit their application. In particular, the injection of viral particles often accompanied by the immune reaction of the body, and direct injection into the target tissue plasmid DNA can provide the expression of angiogenesis inhibitors is usually not more than 1% of tumor cells.

In this regard, a very promising direction in the field of gene therapy of pathological angiogenesis is the use of cells with the previously entered inhibitor gene whose effectiveness has been proved in experiments is about the introduction of experimental animals cultured fibroblasts (Hisaka Y., et al., 2004), genetically transformed skeletal cultured myoblasts (Suzuki K., et al., 2001) and precursors of endothelial cells (Iwaguro h, et al., 2002).

It is obvious that the development of a method that would provide effective local suppression of angiogenesis, in particular, when the growth of tumors, remains a challenge in modern biology and medicine. One of the promising directions in the solution is the unification of the efforts to search for new inhibitors using modern methodological approaches, based on recombinant DNA technology.

Disclosure of inventions

The developed method local suppression of angiogenesis, which consists in ensuring stable target tissue an effective amount of functionally active T-cadherin. The proposed method is based on the established us property cells actively expressing on their surface T-cadherin to inhibit the sprouting of blood vessels in tissues containing these T-cadherin positive cells. Such cells can be as actual cells of the target tissue, and cells that are compatible with the cells of this tissue embedded in it from the outside.

T-cadherin - atypical member of the family of catherinew. Catherine - family of membrane receptors, mediating CA2+dependent homophilous adhesion in all solid tissues of the adult organism and in embryogenesis (Angst et al., 2001). The extracellular part of T-cadherin homologous to corresponding areas of the "classical" representatives of the family and consists of five Ca2+binding cateringowych domains. A unique feature of the T-cadherin is the lack transmembrane and cytoplasmic domains. T-cadherin is fixed on the membrane using glycosylphosphatidylinositol (GPI) "anchors" (Ranscht B, baskets, troubadours-Zimmermann MT., 1991).

Properties of T-cadherin have been actively studied, and to date in this area has accumulated a large amount of factual material. However, the experimental data are rather controversial and not yet allow us to give a definite answer to the question about the basic functions of T-Katherina.

There is evidence that T-cadherin has a weakly pronounced ability to mediate CA2+dependent homophilous cell adhesion in suspension, but due to the lack transmembrane and cytoplasmic domain and connection with the cytoskeleton (Vestal DJ, Ranscht B. 1992) stable intercellular adhesion can hardly be regarded as its primary function. Moreover, the recently obtained experimental data, in accordance with which the overexpression of T-cadherin in cell culture lines NECK and L929 leading to increased cell migration (Rubina K, et al., 2005), and overexpression of T-cadherin in endothelial CL is located, cultivated on a substrate containing a soluble T-cadherin or antibodies to the N-terminal domain of the T-cadherin, causes reduction of cell adhesion to the substrate, the increase in cell migration in the chamber of Boyden and changes in the actin cytoskeleton (Inanov D, et al., 2004;. Philippova M, et al., 2003). These data are most consistent with the notion of T-cadherin as a signaling molecule that regulates cell adhesion and migration.

Information about the expression and the effects of T-cadherin in tumors is also ambiguous and do not provide any definite opinion regarding running T-Katherina functions.

In the study of metastasis carcinoma, Lewis lung has been shown that T-cadherin actively expressed in the endothelium of blood vessels that grow into the tumor, while in the surrounding tissues its expression is not detected (Wyder L, et al., 2002). In normal skin T-cadherin is expressed in the basal layer of the epidermis, hair follicles and sebaceous and sweat glands (Zhou et al., 2002), whereas in samples of various tumors basal cell skin cancer, squamous carcinoma of the skin, colorectal cancer, intestinal expression of T-cadherin in tumor cells reduced (Takeuchi T, et al., 2002; Takeuchi T, et al., 2002; Hibi K, et al., 2003). These data allow us to assume that T-cadherin is a factor, the overwhelming growth and m is testirovanie tumor cells in many types of tumors: breast cancer, lung cancer, ovarian cancer, and basal cell carcinoma of the skin (Takeuchi T, et al., 2002; Yap AS and Kovacs EM, 2003). At the same time on osteosarcoma man was shown that the expression of T-cadherin correlates with tumour metastasis (Takeuchi T and Ohtsuki Y, 2001).

Previously we established that the normal maximum levels of expression of T-cadherin is observed in the nervous and cardiovascular system (cardiomyocytes, endothelium, smooth muscle cells (MMC) and pricetag) (Ivanov et al., 2001). It is known that in the embryonic brain of Chicks T-cadherin performs the function of the molecule-Navigator guiding growing axons of motoneurons (Ranscht B., baskets, troubadours-Zimmermann M.T., 1991; Fredette and Ranscht, 1994), and in culture neurons in vitro inhibits axonal growth (Fredette BJ, et al., 1996).

Our data on the expression of T-cadherin in atherosclerotic lesions of blood vessels, accompanied by changes in cell adhesion, migration and proliferation, the coincidence of the maximum of the expression of T-cadherin in the formation neointima with the peak of the migration and proliferation of cells, an established model of experimental balloon angioplasty of the carotid artery of the rat (Kudrjashova et al., 2002), and some other facts indicate the important role of T-cadherin in the cardiovascular system, which presumably is associated with the regulation of growth and remodeling of blood vessels.

In the process of creating this is about the invention was first experimentally demonstrated the negative impact expressed on the surface of normal cells T-cadherin in the formation of new blood vessels in containing such cells, tissue, that was the basis of the developed method the local suppression of angiogenesis. Due to the fact that the effect is achieved by the expression of T-cadherin as a matter of fact in the cells of the target tissue, and is compatible with this fabric cells introduced into it from the outside, there are two possible alternative embodiments of the method according to the invention.

The proposed method is carried out by introducing into the target tissue or vector suitable for expression contained in the sequence of DNA which encodes a T-cadherin or transformed by the specified vector cells, compatible with the cells of the target tissue. As a DNA sequence that encodes a T-cadherin, can be used the sequence of a gene of the T-cadherin (cadherin-13) man presented in GenBank under number L34058, or other nucleotide sequence encoding the amino acid sequence of the T-cadherin of the person represented in GenBank under AAA 35624.1, and any of their homologues and variants encoding functionally active T-cadherin. Coding T-cadherin DNA sequence can be obtained well-known specialist methods, in particular by cloning the corresponding fragment from cDNA or genomic libraries, PCR amplification using specific primers or chemical C is tesam.

For introduction into the target tissue obtained by any of these methods, the DNA fragment includes expressing in a suitable vector from among well-known specialists vectors, functionally active in mammalian cells. Such a vector may be an adenoviral nature, however, preferred is the use of plasmids, which includes a regulatory region necessary for the expression of T-cadherin in the target tissue. In particular, in one of specific embodiments of the present invention was used plasmid pcDNA 3.1 (Invitrogen) entered into the genome of T-cadherin man, successfully tested earlier with a view to its expression in various cells (Philippova et al., 2003).

In the implementation of the proposed method vector design with the genome of T-cadherin can be directly introduced by injection into the protected region of tissue. However, as practice shows, due to the relatively low efficiency of the delivery of genetic material into cells of the target tissue (the efficiency of transfection of the target tissue when injected plasmids in the fabric is less than 1%) this option requires significant amounts of plasmid DNA to achieve inhibitory concentration of T-cadherin in the target tissue. In light of this, the preferred option seems to be introducing into the target tissue cells, transfirieran the s vector for the expression of T-cadherin and been selected on the basis of its products. When selecting host cells should be considered a condition of their "compatibility" with the cells of the target tissue, which in the present invention was controlled by analysis of cell viability within 2 weeks after injection into the animal organism. We found that for this purpose apply a variety of cell types that are compatible with the cells of the target tissue (in one of the embodiments of the invention in connective tissue was injected cells embryonic kidney, "survival" which after 2 weeks was 10%). However, from the point of view of the obtained effect of inhibition of angiogenesis best results are achieved with the introduction of cells of the same type as the cells of the target tissue (e.g., use case transfected fibroblasts for the introduction of connective tissue).

The number of input vector or input cells should be such to create in the protected area the concentration of T-cadherin, which is sufficient to obtain the effect of inhibiting the growth of new blood vessels. Called quantitative indicators will depend on many factors, including the effectiveness of the expression defined by the type of vector and host cell type of the target tissue, the size of which is protected area, etc. and in each case must be determined empirically.

Thus, the object on which the present invention is a method local suppression of angiogenesis in the target tissue, bringing the target tissue an effective amount of functionally active T-cadherin by introducing a suitable expression vector which includes a DNA sequence encoding a T-cadherin or compatible with target-tissue cells, previously transfected with the indicated vector and expressing the T-cadherin.

Following are specific examples that confirm the possibility of carrying out the invention but do not limit its scope.

Brief description of drawings

Figure 1. Expression of T-cadherin in the selected positive clones determined in the lysates of cells by the method of Western blot turns.

Track 1 - HUVEC cells (positive control), normally Express a Mature T-cadherin (mol. the mass of 105 kDa) and its predecessor (mol mass of 130 kDa).

Track 2 - cells of the control clone (K1) line L929, does not Express T-cadherin;

Track 3 - cell prototype clone (TS) line L929, transfetsirovannyh cDNA that encodes a gene of the T-cadherin; Express a Mature T-cadherin his predecessor.

Track 4 - cell control clone HEK293GF; does not Express T-cadherin.

Track 5 - cell prototype clone (Th5) line NECK, transfetsirovannyh cDNA that encodes a gene of the T-cadherin; Express a Mature T-cadherin his predecessor.

Track 6 - cell prototype clone (Th8) line NECK, transfetsirovannyh cDNA that encodes a gene of the T-cadherin; Express a Mature T-cadherin his predecessor.

Figure 2. Lots of plaques of Matrigel 2 weeks after implantation in NUDE mice.

And - with the control cell line L929, clone K1); B - with experienced cell line L929, clone TS). The differences are statistically significant (p<0,01).

Figure 3. The level of hemoglobin in the plaques of Matrigel 2 weeks after implantation in NUDE mice. Hemoglobin was estimated by the ratio of the absorbance of a solution of gemland (594 nm) to the sample mass (in units of optical density per gram).

And - with the control cell line L929, clone K1); B - with experienced cell line L929, clone TS). The differences are statistically significant (p<0,01).

Figure 4. The number of small capillaries in the plaques of Matrigel 2 weeks after implantation in NUDE mice. Counts the number of vessels without a gap and a length of less than 20 μm and was normalized to the area of the field of view (N).

And - with the control cell line L929, clone K1); B - with experienced cell line L929, clone TS).

The difference being statistically significant (p<0,0001, t-student test).

Figure 5. The number of vessels of medium size in the plaques of Matrigel 2 weeks after implantation in NUDE mice. Counts the number of vessels with lumen or length of 20 μm to 40 μm, were normalized to the size of the field of view (N).

And with the checkpoints for important locations the mi cell line L929, clone K1); B - with experienced cell line L929, clone TS).

The difference being statistically significant (p<0,0001, t-student test).

6. The number of large vessels in the plaques of Matrigel 2 weeks after implantation in NUDE mice. Counts the number of vessels with a diameter of more than 40 μm and were normalized to the size of the field of view (N).

No difference was significantly (p>0,05, U-test Mann-Whitney).

7. Identification of vessels by the method of double immunofluorescent staining using antibodies against D31 on sections of the plaques of Matrigel 2 weeks after implantation in NUDE mice.

And - with the control cell line L929, clone K1); B - with experienced cell line L929, clone TS).

The implementation of the invention

The experiments were conducted on mice lines BULB and NUDE supplied by bioclinical RK SPC MOH and bioclinical Russian Oncological Center, respectively.

To simulate the process of angiogenesis using the method of implantation of experimental animals of Matrigel (Passaniti A, et al., 1982).

Example 1. Obtaining cell clones stably expressing T-cadherin.

In this work, we used cells of the epithelium of the kidney of a human embryo (line HEK 293) and an immortalized mouse fibroblasts (line L929). Cells were grown in CO2- incubator (5% CO2; 95% air) in the medium Needle, modified, Dulbecco (Dulbecco''s Modifed Eagle's Medium, DMEM, GIBCO BRL)containing 10% FBS (fetal bovine serum, HyClone), 100 U/ml penicillin and 10 μg/ml of streptomycin (GIBCO BRL). The change of environment conducted every 2-3 days; reaching the monolayer cells were passively.

Transfection of the cell line HEK293. Cells were subjected to transfection in two stages, using transfection agent Lipofectamin 2000 (Invitrogen), and selected positive clones in a medium containing the selective antibiotic. First, cells were transliterowany the plasmid pEGFP-C1 (Clontech), containing the gene for green fluorescent protein (GFP) gene and the resistance to geneticin (G418) (GIBCO BRL). Cells were selected on medium with geneticin and expressing GFP (HEK293GFP), next was transfusional two plasmids: the plasmid pcDNA 3.1 (Invitrogen) with included genome of T-cadherin person (Philippova et al., 2003) and plasmid pIRESI puro (Clontech), containing the gene for resistance to puromycin. Transfetsirovannyh GFP-positive cells were selected for resistance to puromycin and were analyzed by the content of T-cadherin. Clones HEK293GFP cells obtained in the first stage transfection and expressing the gene of the green fluorescent protein (GFP)was used in further work in quality control. The stability of the constitutive expression of GFP was assessed by fluorescence of the cells in the green channel of the microscope Axiovert 200M, Carl Zeiss, Germany.

Transfection of the cell line L929. Cell line L929 was transfusional plasmas the DOI pcDNA 3.1 genome of T-cadherin (Philippova et al., 2003), using transfection agent Lipofectamin 2000, and selected positive clones in a medium containing the selective antibiotic geneticin, which were analyzed by the content of T-cadherin.

To get the control clones cell line L929 was transfusional the plasmid pcDNA 3.1 (Invitrogen)containing a fragment of the luciferase cDNA in the antisense orientation and the gene of resistance to geneticin (Philippova et al., 2003). For the selection of the control clone cells were incubated in medium with geneticin. The expression of T-cadherin in HEK293 cells (clones h5 and Th8) and L929 (clone TC3) was determined by the method of Western blot turns.

Example 2. Analysis of the expression of T-cadherin in transfected cells.

Preparation of cell lysates. All procedures were performed on ice. Cells growing in bottles for culturing, cooled for 30 min, then washed with cold PBS, scraped off with a plastic spatula and centrifuged at 1500-2000 rpm at +4aboutC for 5 min. the Precipitate was dissolved in lyse buffer (100 mm Tris-HCl, pH 8.1, 1% Triton X-100, 20 mm HEPES (pH 7.5), 150 mm NaCl, 1 mm MgCl2, 1 mm CaCl2inhibitor cocktail of protease in a dilution of 1:100 (Proteinase Inhibitor Cocktail (PIERCE), incubated 15 min, centrifuged for 20 min at 16000 rpm and a temperature of 4aboutC. the Lysate was passed through a syringe with needle G21 for the destruction of polymer chains of DNA, we measured the protein concentration, using the Bio-Rad Protein Assay (Bio-Rad Labortories) and BSA as standard. The resulting lysates were used for electrophoresis of proteins and Western blot turns or kept at -20aboutC.

Electrophoresis of proteins and Western blot turns. Electrophoresis of proteins was performed under denaturing conditions in the presence of SDS. The analyzed sample was mixed with sample buffer (buffer Laemmli (Laemmli)? β-mercaptoethanol), heated to 99aboutC for 5 minutes and put on a 7% SDS page (polyacrylamide gel) of 30 µg protein per well. To determine the molecular masses of the proteins used standard commercial mixture prekrastnyh proteins (Kaleidoscope Prestained Standards, BioRad Laboratories). Transfer of proteins from gel to nitrocellulose membrane (Immobilon, Millipore), pre-moistened with buffer elektroprenos, was carried out by the method of semi electroblotting.

After transfer of proteins the membrane was washed in TBS buffer (150 mm NaCl, 50 mm Tris/HCl, pH 7.4) and incubated in TBS buffer containing 5% skim milk and 0.05% Tween 20 for 60 minutes Next, the membrane was incubated over night at +4aboutWith rabbit antibodies against human T-cadherin previously obtained in our laboratory (Tkachuk et al., 1998). Then the membrane was washed in TBS and incubated in the solution of the second antibody conjugated with horseradish peroxidase (Immune Jackson Research), antibodies against rabbit within 60 minutes. For visualization of protein bands, bound peroxidase antibodies, COI is litovali two-ECL-system (ECL TMWestern Blotting Detection Reagents, Amersham Bioscience); the membrane was exposed to x-ray film CL-XposureTM(Amersham Bioscience).

As a positive control was used endothelial cells of the umbilical vein of a person (HuVEC), which normally Express T-cadherin in Mature form (mol. the mass of 105 kDa) and predecessor (mol. mass of 130 kDa).

According to the results of immunoblotting were selected two clones transfected cell line SOME 293 (h5 and Th8) and one clone transfected cell line L929 (TS) with the highest level of expression of T-cadherin (figure 1). In future work, we used the clone h5 and clone TS.

Example 3. Subcutaneous implantation of Matrigel and cells.

Cells expressing T-cadherin, or control cells were injected into mice subcutaneously in the form of a suspension in Matrigel. For this, cells have reached 90-95% of the density of the monolayer, was removed from the culture cups of 0.25% solution of trypsin in solution Verena, was centrifuged at 1000g for 5 minutes and resuspendable in a small volume of medium; number of cells counted in the camera Goryaeva. All manipulations with cells was carried out in sterile conditions. Then prepared cell suspension in the environment so that in 100 μl medium were 1.7 million cells. Next, 400 μl of cold (+4°C) Matrigel was mixed with 100 ál of cell suspension was injected subcutaneously to mice. In the right side was injected suspension CL is current, do overexpress T-cadherin (experience); in the left side of the suspension control cells. Injections were performed with an insulin syringe with 22G needle thickness so that the Matrigel was able to polymerization under the skin and form a thick, jelly-like implant.

Mice of BALB C as the experimental cells were injected cells HEK293GFP clone Th5, mice NUDE - cell clone TS; as control cells to mice of BALB C was injected control cells HEK293GFP and NUDE mice cells control clone K1.

2 weeks after subcutaneous injection of cells in Matrigel mice were killed isoflurane anesthesia (Isofluran, Fso FloTMmade skin incision over the area of the plaques of Matrigel was removed Matrigel completely and weighed. Next plaques of Matrigel was used to measure hemoglobin or were frozen in liquid nitrogen for subsequent cooking of the slices, immunofluorescence staining and counting of vessels or used for separation of cells from Matrigel to assess their survival.

Example 4. The selection of cells from implants of Matrigel. Selected plaques were crushed in a small volume of sterile PBS (137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4*2H2O, 1.4 mm KH2PO4), was then added to the solution disposi 40 IU/1 ml collagenase 200 IU/1 ml of sterile PBS in a 1:1 ratio and incubated in techenie minutes at 37 aboutWith periodic stirring. After incubation, the enzymes iactiveaware by adding 2 ml of 10% FBS. Cells were besieged by centrifugation at 1000g for 10 minutes. Then the precipitate resuspendable in 1.5 ml buffer for lysis of erythrocytes (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA), incubated for 5 minutes, iactiveaware the addition of 1.5 ml of 10% FBS and centrifuged at 1000g for 10 minutes. Sediment resuspendable in 1 ml of medium DMEM/10% FBS was passed through a 40-μm mesh (BD Biosciences) and were planted on a Petri dish with a diameter of 60 mm cell Survival in Matrigel was evaluated the next day, after separating them from the Matrigel (2 weeks after injection) the ratio of the number of fluorescent cells in the Petri dish to the number entered in the Matrigel cells. Thus were obtained the following results: BALB C/HEK293GF - 10%; BALB C/HEK293GF clone Th5 - 10%; NUDE/ L929 clone K1 - 60%; NUDE/clone L929 TS - 63%.

Example 5. Measurement of hemoglobin in the plaque of Matrigel gentianales method. For measurement of hemoglobin plaques of Matrigel homogenized on electric homogenizer (Janke &Kunkel, IKA-Labor Technic) in 200 μl of 0.9% NaCl and centrifuged for 7 min at 5000 rpm. To the obtained supernatant was added chloroform at the rate of 100 μl of 50 μg of the original sample mass, thoroughly mixed and centrifuged for 3 minutes at 13,000 rpm. To the supernatant was added to 150 μl of hemolytica - concentrated solution Drabkina (K 3Fe(CN)6, KCN, KH2PO4, Triton X-100) and incubated for 15 minutes at room temperature, then centrifuged for 2 min at 13,000 rpm. The resulting supernatant was brought to 0.5 ml of 0.9% NaCl and the resulting solution gemland measured absorption at a wavelength of 594 nm on a spectrophotometer (Janako). The data obtained was normalized according to the standard solution of hemolytica (150 μl of hemolytica +350 ál 0.9% NaCl).

Example 6. Staining of sections plaques of Matrigel method immunofluorescence and counting the number of capillaries.

Plaques were immersed in the medium for frozen tissue Tissue-Tek (Sacura Finetechnical Co., Ltd.), was frozen in liquid nitrogen and kept at -20°or prepared slices of a thickness of 7 μm on a cryostat (NM 505 E., Microm, Germany). Before staining, the slices were fixed in 4% formalin (Sigma) for 10 min at room temperature, then treated with 0.2% Triton X100 for 10 minutes. Then the sections were washed in PBS and incubated in 0,05M solution of ammonium acetate in PBS (10 minutes) to reduce autofluorescence and 1% solution of BSA (bovine serum albumin)containing 10% serum of the donor of the second antibody for driving nonspecific binding (60 min). Further cuts inflicted antibodies rats against mouse CD31, a specific marker of endothelial cells of blood vessels, for 60 minutes. After washing in PBS the sections were placed in a solution of the second ant the phone, conjugated with the fluorochrome Alexa 595 (Molecular Probes) for 60 min For visualization of cell nuclei slices they finished painting areas fluorescente by DAPI. After washing in PBS the sections were concluded on Wednesday Mounting Medium VectashieldTM(Vector Laboratories Inc.). Finished products were stored at +4aboutWith in the dark. Received drugs (Fig.7) were analyzed with a fluorescence microscope Axiovert 200 M (Zeiss). Documentation of images made with a digital camera Axiocam HRC (Zeiss, Germany) and image processing in the program Axiovision 3.1.

Counting vessels held on 4-5 slices from each sample of Matrigel in 4-5 fields of view for each slice, highlighting the capillaries (CD31-positive education without a gap or length less than 20 μm), vessels average diameter (CD31-positive education lumen or longer than 20 µm) and large vessels (diameter > 40 mm). The number of vessels in the same field of view was normalized to the square of the field of vision, assessed by staining of nuclei using DAPI. Counting vessels was performed using the zoom lens 10.

Example 7. Statistical analysis and evaluation of results. Statistical analysis of data was carried out in the program Statistica 6.0. When assessing the weight of the implants and counting the number of vessels used t-student test. The hemoglobin content in the samples was evaluated using the U-test is Anna Whitney. When confirming the normality of the distribution of the characteristic methods of Kolmogorov-Smirnov and Shapiro-Wilkson used the t-student test, for small groups and abnormal distributions - U-test Mann-Whitney.

In both model systems in General obtained similar evidence of suppression under conditions of overexpression of this gene T-cadherin of the process of formation of new blood vessels in the area of implants. As a result of statistical analysis of Matrigel implants with cells experienced clone TS and control clone K1 NUDE mice statistically significant reduction in all key indicators. The total mass of Matrigel implants containing T-cadherin (2, B), more than 1.5 times lower than the mass of the control implants (figure 2, a). The hemoglobin content, normalized by the mass of plaques in experimental implants (Fig 3, B) 3 times less than in control (Fig 3, A). The number of small vessels (capillaries and blood vessels average diameter), normalized by the number of cells stained with nuclear dye DAPI, slice plaques of Matrigel (N in figure 4 and figure 5) in the experimental implants (B) was significantly lower than in the control (A), in particular the number of capillaries in the implants with cells experienced clone TS (figure 4, B) reduced by more than 2 times in comparison with implants containing control cells clone K1 figure 4, A). The number of vessels of large diameter did not depend on the content of T-cadherin in the implant (6).

References

1. Kiselev S.M., Lutsenko S., Severin S.E., E.S. Severin, Inhibitors of tumor angiogenesis. Biochemistry. 2003;68(5): 611-631.

2. Angst B.D., Marcozzi, C., Magee, A.I. The cadherin superfamily: diversity in form and function. J. Cell. Sci. 114: 629-641. 2001.

3. Brantley-Sieders D.M., Chen J. Eph receptor tyrosine kinases in angiogenesis: from development to disease. Angiogenesis. 2004;7(1):17-28.

4. Collett G.D., Canfield, A.E. Angiogenesis and pericytes in the initiation of making emergency ectopic calcification. Circ Res. 2005; 96(9): 930-8.

5. Fischer-Colbrie R., Kirchmair R., Kahler C.M., C.J. Wiedermann, Saria A. Secretoneurin: a new player in angiogenesis and chemotaxis linking nerves, blood vessels and the immune system. Curr Protein Pept Sci. 2005;6(4):373-85.

6. Fredette B.J., Ranscht B. T-cadherin expression delineates specific regions of the developing motor axon-hindlimb projection pathway. J Neurosci. 1994; 14(12): 7331-46.

7. Fredette B.J., Miller J., Ranscht B. Inhibition of motor axon growth by T-cadherin substrata. Development 122(10): 3163-71. 1996.

8. Hibi, K., Nakayama H., Kodera K., Ito K, Akiyama s, Nakao A. CDH13 promoter region is specifically methylated in poorly differentiated colorectal cancer. Brit. J. Cancer. 90: 1030-1033. 2004.

9. Hisaka Y., Ieda M., Nakamura, T., Kosai K., Ogawa, S., Fukuda K. Powerful and controllable angiogenesis by using gene-modified cells expressing human hepatocyte growth factor and thymidine kinase. J Am Coll Cardiol. 2004; 43(10):1915-22.

10. Ivanov D., M. Philippova, Antropova J., Gubaeva F., Iljinskaya O., Tararak E., V. Bochkov, Erne p, Resink T., Tkachuk V. Expression of cell adhesion molecule T-cadherin in the human vasculature. Histochem Cell Biol. 2001; 115(3): 231-42.

11. Inanov D., Philippova, M., V. Tkachuk, Erne p, Resink T. Cell adhesion molecule T-cadherin regulates vascular cell adhesion, phenotype and motility. Exp.Cell Res. 293: 207-218. 2004.

12. Iwaguro H, Yamaguchi J.i., Kalka C., Murosawa, S., Masuda, H., Hayashi, .i., Silver M., Li, T., J.M. Isner, Takayuki A. Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation. 2002; 105: 732-738.

13. Kudrjashova E., Bashtrikov P., Bochkov V, Parfyonova Y., V. Tkachuk, Antropova J., Iljinskaya O., Tararak E., Erne P., Ivanov D., Philippova m, Resink TJ. Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis. Histochem Cell Biol. 2002; 118(4): 281-90.

14. Leong T. T., Fearon u, Veale D.J. Angiogenesis in psoriasis and psoriatic arthritis: clues to disease pathogenesis. Curr Rheumatol Rep. 2005; 7(4): 325-9.

15. Pap, T., O. Distler Linking angiogenesis to bone destruction in arthritis. Arthritis Rheum. 2005; 52(5): 1346-8.

16. Passaniti, A., Taylor R., Pili R., Guo Y., Long, P.V., Haney J.A., et al. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest 1982; 67:519-528.

17. Philippova, M., D. Ivanov, V. Tkachuk, Erne P. and Resink TJ: Polarisation of T-cadherin to the leading edge of migrating vascular cells in vitro: a function in vascular cell motility? Histochem Cell Biol 120: 353-360, 2003.

18. Ponnazhagan, S., Mahendra G., Kumar, S., Shaw D.R., Stockard C.R., Grizzle W.E., S. Meleth Adeno-associated virus 2-mediated antiangiogenic cancer gene therapy: long-term efficacy of a vector encoding angiostatin and endostatin over vectors encoding a single factor. Cancer Res.; 64(5): 1781-7, 2004.

19. Ranscht B., baskets, troubadours-Zimmermann M.T. T-cadherin, a novel cadherin cell adhesion molecule in the nervous system lacks the conserved cytoplasmic region. Neuron 7(3): 391-402. 1991.

20. Rubina K., Talovskaya K., Cherenkov V., Ivanov D., Stambolsky D., Storozhevikh T., Pinelis V., A. Shevelev, Parfyonova Ye., Resink, T., Erne P., Tkachuk V. LDL dosage intracellular signaling and cell migration via atypical LDL-binding protein T-cadherin. Mol. Cell. Biochemistry. 373: 33-41. 2005.

21. Seth P. Vector-mediated cancer gene therapy: an overview. Cancer Biol Ther. 4(5):512-7. 2005.

22. Suzuki K., Brand N.J., Allen, S., M.A. Khan, Farrell A.O., Murtuza b, Oakley R.E., Yacoub M.H. Overexpression of connexin 43 in skeletal myoblasts: Relevance to cell tansplantation to the heart. J.Thorac Cardiovasc Surg. 122:759-766. 2001.

23. Takeuchi, T., Ohtsuki Y. Recent progress in T-cadherin (CDH13, H-cadherin) research. Histol. Histopathol. 16: 1287-1293. 2001.

24. Takeuchi, T., Liang, S.-B., Ohtsuki Y. Downregulation of expression of a novel cadherin molecule, T-cadherin, in basal cell carcinoma of the skin. Mol. Cancerogen. 35: 173-179. 2002.

25. Takeuchi, T., Liang, S.-B., Matsuyoshi, N., Zhou, S., Miyachi Y., Sonobe h, Ohtsuki Y. Loss of T-cadherin (CDH13, H-cadherin) expression in cutaneous squamous cell carcinoma. Lab. Invest. 82(8): 1023-1029. 2002

26. Tkachuk VA, V.N. Bochkov, Philippova M.P., Stambolsky DV, Kuzmenko E.S., Sidorova, M.V., Molokoedov A.S., Spirov V.G. and Resink TJ: Identification of an atypical lipoprotein-binding protein from human aortic smooth muscle as T-cadherin. FEBS Lett 421: 208-12., 1998.

27. Vestal D.J., Ranscht B. Glycosyl phosphatidylinositol-dialog T-cadherin mediates calcium-dependent, homophilic cell adhesion. J Cell Biol. 119: 451-61. 1992.

28. Wyder L., Vitality, A., Schneider H., Hebbrand L.W., Moritz D.R., M. Wittmer, M. Ajmo, Klemenz R. Increased expression of H/T-cadherin in tumor-penetrating vessels. Cancer Res. 60: 4682-4688. 2000.

29. Zhou, S., N. Matsuyoshi, Liang S.-B., Takeuchi t, Ohtsuki y, Miyachi Y. Expression of T-cadherin in basal kerationcytes of skin. J. Invest.Derm. 118(6):1080-1096. 2002.

30. Yap A.S., Kovacs EM Direct cadherin-activated cell signaling: a view from the plasma membrane. J. Cell. Biol. 160: 11-16. 2003.

1. The way the local suppression of angiogenesis in the target tissue, bringing the target tissue an effective amount of functionally active T-cadherin by introducing into it a suitable expression vector which includes a DNA sequence encoding a T-cadherin or compatible with target-tissue cells, previously transfected with the indicated vector and expressing the T-cadherin.

2. The method according to claim 1, featuring the the action scene, they use the expression vector, which is a plasmid.

3. The method according to claim 2, characterized in that use plasmid pcDNA 3.1.

4. The method according to claim 1, characterized in that the protected area (the target tissue) to introduce a suitable expression vector, which includes encoding a T-cadherin DNA sequence.

5. The method according to claim 1, characterized in that the target tissue is injected compatible cells, pre-transfetsirovannyh a suitable expression vector, which includes encoding a T-cadherin DNA sequence and expressing the T-cadherin.

6. The method according to claim 5, characterized in that is compatible with the target-tissue cells are cells of the same type as the cells of the target tissue.

7. The method according to claim 6, characterized in that the target-tissue is connective tissue, and the input cells are fibroblasts.



 

Same patents:

FIELD: immunology, biotechnology.

SUBSTANCE: described are rabies virus-neutralizing antibody and fragments thereof; method for treatment of subject being affected by rabies virus using said antibody and fragments thereof. Disclosed are variants of isolated nucleic acids, encoding polypeptides bearing light and/or heavy antibody strain, respectively. Disclosed is expression vector bearing at least one from abovementioned nucleic acids. The invention makes it possible to increase subject survival after rabies virus attack.

EFFECT: method for prophylaxis and treatment of rabies virus infections.

14 cl, 1 tbl, 5 ex

FIELD: biotechnology.

SUBSTANCE: claimed method includes injection of vector construct pX-RSVhGH encoding human growth hormone or pX-Ins, encoding human insulin into parenchyma of 8-9-week rabbit males. Transformation effectiveness of gene constructs is determined by immunohystochemical kit Novocastra (Sigma, USA).

EFFECT: method for genetic transformation of rabbit testicle stem cells with increased effectiveness.

3 dwg, 1 ex

FIELD: molecular biology, veterinary.

SUBSTANCE: invention proposes isolated DNA sequence (variants) encoding Ehrlichia canis protein of size 30 kDa. Also, invention proposes vector comprising such sequence, recombinant Ehrlichia canis 28 kDa protein encoded by this sequence, a cell-host comprising this sequence, a method for preparing the protein, immunoreactive antibody specific to this protein and a method for inhibition of Ehrlichia canis infection in subject. Recombinant protein of size 28 kDa from Ehrlichia canis shows immune reactivity with respect to serum against Ehrlichia canis. Proposed group of inventions can be used in development of vaccines and serodiagnosticum that shows high effectiveness for prophylaxis of diseases and for carrying out the serodiagnosis.

EFFECT: improved preparing method, valuable medicinal and veterinary properties of protein.

19 cl, 17 dwg, 8 ex

FIELD: biotechnology, biochemistry, microbiology.

SUBSTANCE: method involves to a method for preparing the fodder complex enzyme preparation possessing activities of endo-1,4-β-glucanase, β-glucanase, endo-1,4-β-xylanase, phytase, pectin-lyase and α-galactosidase. The preparation is prepared by combined culturing multicopy strains of fungus Penicillium canescens VKM F-3868D and VKM F-3869D. The enzyme preparation possessing activities of endo-1,4-β-glucanase, β-glucanase, endo-1,4-β-xylanase, phytase and pectin-lyase is prepared in combined culturing multicopy strains of fungus Penicillium canescens VKM F-3868D and VKM F-3870D. For preparing the enzyme preparation possessing activities of endo-1,4-β-glucanase and β-glucanase the strain Penicillium canescens VKM F-3868D is cultured, and for preparing the enzyme preparation possessing activities of phytase, pectin-lyase and α-galactosidase the strain Penicillium canescens VKM F-3869D is cultured. The enzyme preparation possessing activities of phytase and pectin-lyase is prepared by culturing the strain Penicillium canescens VKM F-3870. The claimed invention provides expanding assortment of enzyme preparations.

EFFECT: improved preparing method.

12 cl, 3 dwg, 12 ex

FIELD: microbiology, genetics of microorganisms.

SUBSTANCE: invention relates to methods for transduction of anthrax pathogen and closely related bacilli. Method for transduction of Bacillus anthracis and closely related bacilli involves incubation a mixture of bacteriophage with recipient cells taken in the definite ratio. After the first bacteriophage generation in 20 min the preparation of recipient cell receptors is added to the transducing system and the transducing system is inoculated on selective media. Invention describes a method for preparing receptors from recipient cells B. anthracis, B. thuringiensis and B. cereus. Using the proposed method provides the full-value substitution of antiphage serum in the transducing system for a cheaper and simple in preparing component, namely, the preparation prepared from recipient cell receptors that neutralizes phage effectively and prevents excessive death of the recipient strain cells in the transducing system.

EFFECT: improved method for transduction.

9 dwg, 1 tbl, 2 ex

FIELD: immunology, biology.

SUBSTANCE: invention relates to variants of IL-1β-binding molecule having common functionally active sites (CDR sites) and may bind human IL-1β. Said molecules have neutralizing activity IC50 of approximately 50 pM and binding constant KD of approximately 30 pM. Amino acid sequence is described in description of present invention. Variants of DNA constructs encoding of heavy chain and light chain of IL-1β-binding molecule are disclosed. Expression vectors carrying at least one abovementioned nuclear acid and method for production of IL-1β-binding molecules by using the same also are described.

EFFECT: IL-1β-binding molecules against human IL-1β with high neutralizing activity and binding constant useful in suppression of HAMA response.

10 cl, 1 dwg, 3 tbl, 4 ex

FIELD: biotechnology, in particular production of modified swine factor VIII (POL1212).

SUBSTANCE: DNA molecule encoding of modified swine factor VIII is cloned in expression vector, having functionality in mammalian cells. Modified swine factor VIII protein is obtained by cultivation of mammalian cell line BHK CRL-1632 (ATCC), BHK 1632, or CHO-K1, transfected with vector. Therapeutic composition for treatment of subjects suffering from deficit of factor VIII, such as haemophilia, contains effective amount of swine factor VIII protein.

EFFECT: effective agent for treatment of factor VIII deficit.

13 cl, 8 dwg, 7 ex

FIELD: biotechnology, medicine.

SUBSTANCE: invention relates to new recombinant allergens that represent mutants of allergens of the natural origin and comprising at least four mutations. Examples of recombinant allergens are allergens Bet v1 and Ves v1. The primary mutations in recombinant allergen are separated of one another by interval for at least 15 Å and is location is characterized by that at least one circle region of surface of size 800 Å doesn't comprise mutations. Recombinant allergens are used as a pharmaceutical agent as a component of pharmaceutical composition that represents vaccine against allergic response reactions. Invention describes methods for using recombinant allergens in pharmaceutical composition for producing the immune response in subject. Invention represents DNA sequences given in the invention claim that encode recombinant allergens, expressing vector comprising DNA and cell-host for providing the recombinant allergen. Also, invention describes methods for preparing pharmaceutical composition and recombinant mutant allergen. Using recombinant allergen allows decreasing the specific IgE-binding capacity as compared with IgE-binding capacity of the natural allergen. Invention can be used in medicine for preparing vaccine against allergic response reactions.

EFFECT: valuable medicinal properties of allergens.

33 cl, 62 dwg, 10 ex

FIELD: biology.

SUBSTANCE: invention relates to nucleotide sequence associated with increasing or reducing of ovulatory rate in mammalians, namely GDF-9B. Mutated GDF-9B molecule useful in modulation of ovulatory rate in female mammalians is disclosed. Also disclosed are various methods for modulation of ovulatory rate and composition for method embodiment.

EFFECT: method for inducing of sterility or reduced fertility of female mammalians.

30 cl, 14 dwg, 6 tbl

FIELD: biotechnology.

SUBSTANCE: invention relates to isolated nucleic acid sequence encoding of polypeptide with nitrilase activity, wherein nitriles are converted to carboxylic acids in presence of said nitrilase.

EFFECT: method for production of chiral carboxylic acids with high effectiveness and low cost.

10 cl, 4 dwg, 2 tbl, 1 ex

FIELD: biochemistry, biotechnology, peptides.

SUBSTANCE: invention relates to protein of molecular mass 12000 Da isolated from medicinal leech saliva (Hirudo medicinalis) comprising 6 cysteine residues able to form bonds -S-S-, and pI value about 3.7. The novel protein possesses ability to block platelets adhesion induced by collagen. As result of screening cDNA library of H. medicinalis a novel gene of new inhibitor of adhesion is identified and its nucleotide sequence is determined. Invention describes a method for preparing recombinant form of protein involving transformation of suitable cells with vector comprising DNA sequence encoding inhibitor, culturing cells under condition providing its expression and isolation of expressed protein. Invention proposes using natural and recombinant forms of inhibitor in therapy states associated with vessels congestion and diseases of circulatory system, and for treatment of articles surface made of natural and artificial collagen.

EFFECT: valuable biological and medicinal properties of polypeptide.

17 cl, 10 dwg, 13 ex

FIELD: pharmaceuticals.

SUBSTANCE: claimed method includes composition containing mannan-binding lectin and treatment of conditions associated with deficit of mannan-binding lectin, causing susceptibility to infective disorders. Also disclosed is method for production of new expression structure encoding human mannan-binding lectin.

EFFECT: method for production of new human mannan-binding lectin.

47 cl, 8 ex, 5 dwg

FIELD: gene engineering, in particular preparation based on PDGF-BB, useful in therapy, veterinary, diagnosis, tissue culturing.

SUBSTANCE: invention relates to PDGF-BB production using Pichia pastoris 2-2 strain as producer of human platelet growth factor. Produced two-dimensional PDGF-BB form is characterized with purity of 95 %, specific activity of 3-5 ED50/ng, and expression level in yeast up to 0.1 g/l of culture.

EFFECT: two-dimensional PDGF-BB form with high purity, specific activity and expression level in yeast.

5 cl, 11 dwg, 5 ex

FIELD: genetic engineering, biotechnology, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to DNA encoding glycoprotein VI (GP VI) used as a base for synthesis of recombinant GP VI by method of recombinant DNAs followed by its using for the development of pharmaceutical compositions possessing with capacity for inhibiting or blocking interaction of platelets with collagen. Using the invention provides preparing GP VI in the recombinant form and using this agent for therapeutic aims.

EFFECT: valuable medicinal properties of glycoprotein VI.

6 cl, 4 dwg, 6 ex

FIELD: biotechnology, gene engineering, medicine.

SUBSTANCE: polypeptide (IL-li) is obtained by recombinant DNA technology having activity of interleukin-1-alpha and interleukin-1-beta inhibitor. Disclosed is DNA sequences encoding inhibitor precursor and maturated form thereof. Described is method for production of vector constructs containing said DNA sequences, as well as transformed cells producing of recombinant IL-li.

EFFECT: method for large-scale production of interleukin-1-beta inhibitor, useful in medicine.

21 cl, 26 dwg, 11 ex

FIELD: biotechnology, in particular production of modified swine factor VIII (POL1212).

SUBSTANCE: DNA molecule encoding of modified swine factor VIII is cloned in expression vector, having functionality in mammalian cells. Modified swine factor VIII protein is obtained by cultivation of mammalian cell line BHK CRL-1632 (ATCC), BHK 1632, or CHO-K1, transfected with vector. Therapeutic composition for treatment of subjects suffering from deficit of factor VIII, such as haemophilia, contains effective amount of swine factor VIII protein.

EFFECT: effective agent for treatment of factor VIII deficit.

13 cl, 8 dwg, 7 ex

FIELD: immunobiotechnology.

SUBSTANCE: invention relates to soluble CTLA4, which represents mutant variant of wild type CTLA4 and conserves binding ability to CD80 and/or CD86. Molecules of soluble CTLA4 have the first amino acid sequence containing extracellular CTLA4 region, which includes some mutant amino acid residues in S25-R33 region and M97-G107 region. According the present invention mutant molecules also may include second amino acid sequence, enhancing solubility of mutant molecule. Nucleic acid (NA) molecules encoding said CTLA4 and including NA-vectors also are described. Invention also relates to method for production of mutant CTLA4 and uses thereof in controlling of interaction between T-cell and CD80 and/or CD86-positive cell; suppression of graft-versus-host reaction; and treatment of immune system diseases. Soluble mutant CTLA4 according to present invention binds to CD80 and/or CD86 antigen with higher avidity than wild type CTLA4 or non-mutant CTLA41g.

EFFECT: new preparation for treatment of immune system diseases.

65 cl, 19 dwg, 2 tbl, 2 ex

FIELD: medicine, biotechnology.

SUBSTANCE: invention relates to antibodies specifically binding to new human extracellular matrix polypeptides called as RGI; immunoconjugate containing the same and method for selective cell degradation; method for treatment of prostates cancer and metastasis in patients suffering from prostates cancer.

EFFECT: new method for treatment of prostates cancer.

28 cl, 7 ex, 7 dwg

FIELD: molecular biology, genetic engineering, polypeptides, medicine.

SUBSTANCE: in using the double-hybrid yeast system DNA sequences encoding polypeptides (55.1 and 55.3) have been found that elicit ability for binding with intracellular domain p-55 (p-55IC) of TNF-receptor. It has been established that these polypeptides represent fragments of amino acid sequences p-55IC, respectively, from 338 to 426 and from 277 to 426 residues. As result of insertion of DNA fragments with a sequence encoding polypeptide 55.1 or 55.3 into the structure of expressing vector and transformation suitable host-cells by this vector recombinant form of indicated polypeptides have been prepared. Using this invention provides the possibility for modulating the function of intact p-55 of TNF-receptor. Invention can be used in medicine in treatment of diseases associated with transfer of TNF-signal.

EFFECT: improved preparing method and valuable properties of polypeptide.

9 cl, 17 dwg, 3 tbl, 6 ex

FIELD: biotechnology, molecular biology, medicine.

SUBSTANCE: invention discloses amino acid sequences of human obesity polypeptide (OB) two isoforms possessing capacity for modulation of animal body mass, their signal peptide-containing precursors and analogues. Polypeptide isoforms are prepared as result of insertions, deletions and amino acid changes that retain activity typical for nonmodified forms of OB-polypeptides, and polyclonal and monoclonal antibodies interaction specifically with new agents modulating the body mass value also. Invention describes DNA sequences encoding these polypeptides and their analogues, vector structures comprising these sequences used for preparing recombinant forms of OB-polypeptides. Invention proposes using new polypeptides and their analogues as an active component in pharmaceutical compositions. Using this invention can promote to solving the problem for providing medicine, veterinary science and animal husbandry with effective agent used for decreasing the body mass value. Invention can be used in medicine for diagnosis and treatment of pathological states associated with disturbance of regulation of human body mass, and in animal husbandry and veterinary science.

EFFECT: valuable biological, medicinal and veterinary properties of polypeptide.

23 cl, 71 dwg, 12 tbl, 17 ex

FIELD: genetic engineering, biotechnology, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to DNA encoding glycoprotein VI (GP VI) used as a base for synthesis of recombinant GP VI by method of recombinant DNAs followed by its using for the development of pharmaceutical compositions possessing with capacity for inhibiting or blocking interaction of platelets with collagen. Using the invention provides preparing GP VI in the recombinant form and using this agent for therapeutic aims.

EFFECT: valuable medicinal properties of glycoprotein VI.

6 cl, 4 dwg, 6 ex

Up!