Method and transplant for treatment of liver failure

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and can be applied for treatment of liver failure. Transplant includes three-dimensional biocompatible biodegradable porous matrix, which has total volume not less than 0.05 cm3 and the smallest linear size not less than 0.2 mm, pore size 2-500 mcm, total porosity 50-97%, and implanted on it allogenic progenitor cells of bone marrow and allogenic liver cells, concentration of liver and bone marrow cells being 2×106-15×106 cells per 1 cm3 of matrix and ratio of bone marrow cells to liver cells being from 1:1 to 1:4. In realisation of method of treating liver failure transplant is placed into mesentery of small intestine.

EFFECT: group of inventions makes it possible to increase term of cell survival, activate their proliferation.

6 cl, 7 dwg

 

The invention relates to medicine, transplantation, namely, cell transplantation, and can be used in the correction and treatment of liver failure. The proposed method and the graft can be used in specialized departments involved in the treatment and correction of liver failure.

There is a method of treatment of liver failure, based on the use of suspensions of isolated xenogeneic hepatocytes in extrakorporale system supporting the liver, through which perpusilla the blood of the recipient liver (Shumakov V.I. and co-authors. Essays on physiological problems of Transplantology and artificial organs. // Tula. - Reprints. - 1998. - s-362).

Isolated hepatocytes in such systems, as xenogenic capable in the body of the patient only for a short time to carry out detoxification and synthetic function. At the same time, the ability of suspensions of isolated hepatocytes to allocate regulatory peptides, stimulates regeneration processes in the diseased liver recipient, extended to 0.5-8 hours (Shumakov V.I. and other Treatment of severe liver failure perfusion of the patient's blood through a suspension of cryopreserved hepatocytes. // Surgery. - 1990. No. 2. - P.113-116).

Know of any other way of treatment, the Oia liver failure, using microfragments liver tissue, which helped to prolong the metabolic activity of hepatocytes contained in microfragments to 24-34 and even up to 48 hours (Solovyev V.V., Onishchenko N.A., Akatov B.C., Lezhnev EI Functional activity of hepatocytes in the slices of liver in vitro: dependence on size of the fragments and the length of their cultivation. // Bull. The experts. Biol. and Honey., - 1997, - №10, - s-408). Term increase in metabolic activity of hepatocytes in the composition of microfragments is due to the saving of intercellular contacts and the natural extracellular matrix, as well as due to the preservation of the spatial structure of the liver tissue, resulting in optimized conditions for the functioning of all cell types, including hepatocytes.

Also known is a method of treatment of hepatic failure involving for prolonging the time of survival of isolated hepatocytes in perfusion bioreactors use of isolated hepatocytes with Mironosetsky (using, for example, cytodex-3 with a ratio of 1.6 g of micronesias 1×109cells), which are pre-coated with collagen (Dimetriou A.A., Rozga J., L. Podesta Early clinical experience with a hybrid bioartificial liver // Scan. J. gastroenterol. - 1995. - 30. - Suppl. 208. - p.111-117; Chen, S., Eguchi S, Watanabe Hepatic support strategies // Transplant. Proc. - 1996. - 28, No. 4. - R-2038).

As proto the IPA we have chosen a method of treatment of liver failure, using isolated hepatocytes for auxiliary support of the liver and, consequently, fill detoxification and bioregulatory functions of the damaged liver (Van de Kerkhove M.P., R. Hoekstra, Chamuleau R.A., van Gulik TM Clinical application of bioartificial liver support systems. // Ann. Surg. - 2004. - Vol.240. - P.216-230).

The disadvantages of known methods, including prototype, predpolagaemyh the use of extracorporeal bioreactor with microfragments liver tissue or isolated hepatocytes include:

- the necessity of using special grinders to obtain fragments of a given size, which increases the risk of infection from donor material;

- the necessity of using perfusion systems and inclusion in the perfusion circuit additional components for oxygenation and detoxification of the blood (plasma)coming to microfragments liver tissue, which increases the economic cost method.

- the need of the use of bioreactors microfragments in a mixture with particles of a porous biocompatible carrier to prevent adhesion of microfragments and ensure the effective mass transfer;

- the use of particles of porous media does not create conditions to prevent delayed death of hepatocytes in microfragments, because with the increase date the cultivation of microfragments due to the low rate of proliferation of hepatocytes begins to form a monolayer of quickly attached and proliferating stromal cells, enveloping local liver tissue and impairing their functioning;

- the necessity of using xenogenic material that increases the risk of transfer of dangerous infections and contributes to excessive activation of the immune system of the recipient with the reduction of the period of operation of this material;

- early termination (1-2 days) metabolic and regulatory functions of hepatocytes;

- the need to regularly exercise sessions connected perfusion systems "bioisostere liver".

A device for the treatment of liver failure in vitro method of connection of isolated hepatocytes (Dimetriou AA, Rozga J., L. Podesta Early clinical experience with a hybrid bioartificial liver // Scan. J. gastroenterol. - 1995. - 30. - Suppl. 208. - p.111-117). Also known is a device designed for the treatment of liver failure, which can increase the period of viability of isolated hepatocytes (Shumakov V.I. and co-authors. Essays on physiological problems of Transplantology and artificial organs // Tula. - Reprints. - 1998. - s-362).

As a prototype of the proposed transplant for the treatment of liver failure we have chosen intracorporeal transplant (bimodal) "auxiliary liver" (Uyama, S., Kaufman, P., Kneser U., Vacanti J., Rodriges X. Hepatocyte transplantation using biodegradable matrices in ascorbic acid-deficient rats: comparsion with hetertropically transplanted liver grafts // Transplantation. - 2001. - Vol.7. - P.1-7).

The disadvantages of the known devices, including prototype, predpolagaemyh using bimodule with isolated hepatocytes include:

- severe inflammatory reaction to the graft;

- the death of a large number of isolated hepatocytes;

- low density attaching cells of the liver;

- high cost matrix carrier.

The objective of the invention is to develop a method that improves the efficiency of correction and treatment of liver failure due to prolongation of the terms of the survival of isolated hepatocytes and their implementation functional and organ-specific activity by transplant long-term functioning intracorporeal graft type "auxiliary liver".

Technical result achieved in the implementation of the invention, consists in the correction and treatment of liver failure by providing isolated hepatocytes detoxification function, prolongation of the terms of their survival by activation of proliferation through joint intracorporeal injection progenitor cells in the bone marrow, create a frame for placement in the space of associations formed cells, creating conditions for germination in this framework vessels, diffusion of nutrients, KIS is Orada and factors of tissue differentiation of the blood vessels of the mesentery and blood vessels, sprouted in the frame, to the immobilized liver cells and bone marrow cells; and in the prevention of complications associated with blood clots and cellular infiltration at the site of transplantation of the graft with the cellular material.

The advantages of the proposed method and graft for the treatment of liver failure, allowing essentially to create intracorporeal system auxiliary liver for long-term maintenance of functional status of isolated hepatocytes are:

- no need to use complex perfusion systems;

- creation using a 3-dimensional matrix of culture conditions for cell proliferation and the formation of a "tissue-like" structure;

- creation of adequate conditions for diffusion oxygenated interstitial fluid and sprouting of blood vessels through the matrix that prolong adequate conditions of life planted cells;

- use allogenic cells allow this system to provide in the body long bioregulatory effects.

In the proposed invention does not use tissue and/or cellular material of human embryos. Used cellular material adult donors.

Co-cultivation of liver cells and bone marrow prior to transplantation allows for an optional stand is to enhance the functional activity of hepatocytes by releasing growth factors and cytokines, secreted by bone marrow cells and creates conditions for the differentiation of bone marrow cells in the endothelial cells to form blood vessels and their germination in the matrix. This contributes to a more rapid integration of the graft into the circulatory system after transplantation and maintain metabolism and vital functions of cells by delivery of the newly educated and sprouted vessels oxygen; creation of adequate conditions for diffusion oxygenated interstitial fluid that prolong adequate conditions of life planted cells.

The use of anticoagulants, antiaggregants and immunosupressants immediately after transplantation inhibits the cellular response to the transplant as foreign body and prevents the formation of thrombi at the site of transplantation and rejection.

The essence of the invention is as follows.

For the treatment of liver failure use of isolated liver cells. Taking allogeneic progenitor bone marrow cells from the donor, then taking allogeneic liver cells from the donor. Perform landing liver cells and bone marrow cells on three-dimensional biocompatible biodegradable porous matrix. The pore size of the matrix varies from 2 to 500 μm. The total porosity of the matrix is 50-97%. The total concentration of cells is echani and bone marrow is 2×10 6-15×106cells on 1 cm3matrix. The ratio of bone marrow cells to liver cells from 1:1 to 1:4. And the total amount of matrix is not less than 0.05 cm3. The smallest linear dimension of the matrix should be not less than 0.2 mm Transplantation matrix with cells of the liver and bone marrow produce in the mesentery of the small intestine.

In the particular case after the landing of the liver cells and bone marrow in the matrix prior to transplantation carry out the co-cultivation of allogenic liver cells and bone marrow cells, for example, in 2-3 days.

In the particular case immediately after transplantation prescribe anticoagulants in the prevention dose. Dose: heparin 2500 ME every 12 hours for 7-10 days under the control of the blood coagulation system and antiplatelet agents in the prevention dose, e.g trental based 45 mg/m2body surface area every 12 hours for 30 to 90 days.

In the particular case immediately after transplantation designate immunosupressant in the preventive dose. Dose: human chorionic gonadotropin 500-5000 ME 1 time per day for 30-90 days.

The proposed transplant for the treatment of liver failure includes a three-dimensional biocompatible biodegradable porous matrix and planted on him allogeneic bone marrow cells and allogeneic liver cells. The total amount of the matrix is not less than 0.05 cm 3and the smallest linear dimension is not less than 0.2 mm pore Size of the matrix 2-500 μm, the total porosity 50-97%. Concentration of liver cells and bone marrow 2×106-15×106cells on 1 cm3matrix and the ratio of bone marrow cells to liver cells from 1:1 to 1:4.

The proposed method and the transplant allow prolongirovanne to correct acute or chronic liver failure due to pregnenolone functioning of isolated hepatocytes. The use of immunosupressants eliminates complications associated with rejection reaction. The proposed group of inventions allows also:

1. To create conditions for the attachment of isolated liver cells and bone marrow cells to a biocompatible matrix, stimulates their proliferation, which is supported by diffusion in the matrix of nutrients, oxygen and factors of tissue differentiation of blood flowing to the matrix of the mesenteric vessels.

2. To create a frame-based biocompatible biodegradable and porous matrices that mimic the spatial structure for attaching cells of the donor liver and progenitor cells from the bone marrow, as well as conditions for germination in this framework vessels feeding the attached cells.

3. To create conditions that prevent cell infiltration Tran is plantat and its supporting cells in a viable state through the use of anticoagulants and antiplatelet agents.

4. To create conditions that prevent graft rejection and its supporting cells in a viable state through the use of immunosuppressants.

The method is as follows and includes several stages:

1). The selection of allogeneic progenitor cells from the bone marrow.

The allocation of progenitor bone marrow cells from the donor are carried out by the traditional method (Shumakov V.I., Onishchenko., Krasheninnikov M.E. Ter-Minassian, and other Bone marrow as a source of mesenchymal cells to restore the treatment of damaged organs. // The Bulletin of transplantation and the suit. bodies. 2002, 4, p.3-6; Shumakov V.I., Onishchenko N.A. et al. Biological reserves bone marrow cells and correction of organ dysfunction. // Moscow, Laurel. 2009. - p.61-67). The dotted line in the iliac bone of the donor and take the bone marrow in the amount of 40-150 ml in a sterile container with Hanks solution containing 200 μg/ml gentamicin; and 10.0 μg/ml insulin; 0.25 μm of dexamethasone; 250 u/ml of heparin. Cell suspension centrifuged 5 min at 1500 rpm and the precipitate cells resuspended in lyse solution (114 mm NH4Cl; 7.5 mm KHCO3; 100 μm EDTA), at a ratio of 1:4 from the original volume of aspirate for 5-10 min and centrifuged 3 min at 1500 rpm at room temperature. Hamalsarany supernatant remove odasi what W. Achieve complete lysis of red blood cells, for which the procedure lizirovania spend three times with subsequent laundering of the cells by centrifugation. Cellular precipitate free of erythroid and platelet forms resuspended in the growth environment.

2). The selection of allogenic liver cells.

Produce resection of 2-4×2-4×1-2 cm of the liver tissue from the donor to obtain liver cells. The selection of allogeneic cells of the liver are carried out by the traditional method (Fontaine M, Schloo B, Jenkins R, Uyama S, Hansen L, Vacanti J.P. Human hepatocyte isolation and transplantation into an athymic rat, using prevascularized cell polymer constructs. // Pediatr. Surg. - 1995. - vol.30(1). - P.56-60; Hang H, Shi X, Gu G, Wu Y, Ding Y. A simple isolation and cryopreservation method for adult human hepatocytes // Int J Artif Organs. 2009 Oct; 32(10). - P.720-7; Lehec S.C, Hughes RD, Mitry R.R, graver carving M.A, Verma A, Wade J.J, if you A. Experience of microbiological screening of human hepatocytes for clinical transplantation. // Cell Transplant. 2009; 18(8). - P.941-947; O. Seglen Preparation of isolated rat liver cells. // Methods. Cell. Biol. 1976, the - vol.13. - P.29-83).

The selection of allogenic liver cells produce of the resected portion of the liver by 3 times washing piece of liver from the blood and grinding it to a cold (t=4°C) in a Petri dish, with 3 times washing the resulting suspension buffer solution without calcium [1000 ml of distilled water, 8.3 g of NaCl, 0.5 g KCl, 2.38 g HEPES, pH 7.4, 37°C] for 7 minutes. After this, small pieces of liver are incubated 3 times with a solution of collagenase [1000 ml of distilled water, 8.3 g of NaCl, 0.5 g KCl, 0.7 g of CaCl2, 2.38 g HEPES, .5 mg trypsin inhibitor and 500 mg of collagenase Type IV-S (> 125 CDU/mg), pH of 7.3; 37°C] for 6-8 minutes, followed by replacing the enzyme solution using centrifugation at 500 rpm for 1 minute at t=37°C. the resulting material was transferred to a sieve with 200 µm and filtered washing with culture medium William's E with 10% fetal bovine serum, after which a suspension of single cells and small aggregates are transferred to a centrifuge tube and centrifuged at 500 rpm at 4°C for 1 minute. The supernatant is removed, the residue resuspended in the same fresh medium and centrifuged again. The procedure was repeated 3 times. Cell viability assessed by the method of dyeing Trifanova blue. Get get the number of cells in the range from 3.0×108to 4.0×10 hepatocytes 12-15 g of liver tissue. The cell suspension should contain: hepatocytes, neverthemore liver cells, which determine when light microscopy. Division prehistoric and neprekonatelnych cells do not perform. A suspension of liver cells concentrate in 1-2 ml of physiological solution.

3). Landing (immobilization) of allogenic liver cells and allogeneic bone marrow cells to the media.

Landing (immobilization) of cellular material is carried out according to traditional methods (D.J. Mooney, Sano K., Kaufmann P.M., Majahod K., Schloo Century, Vacanti JP, Langer R. Long-term engraftment of hepatocytes transplanted on biodegradable polymer sponges // J. Biomed. Maer. Res, - 1997. - Vol.5. - P.413-420).

Allogeneic liver cells and allogenic progenitor cells in the bone marrow resuspendable in the growth medium [William's E with the substitution of arginine to ornithine, with the addition of fetal bovine serum, growth factor hepatocytes, epidermal growth factor, β-subunit of the cholera toxin, dexamethasone, ethanolamine, sodium Selenite, glucagon, insulin, insulin-like growth factor-1, ascorbic acid, linolevoi and linelevel fatty acids] in a concentration of 2.0 to 4.0×106liver cells/ml and 2,0-4,0×106progenitor bone marrow cells/ml Total cell suspension was applied to the matrix at a concentration of 2×106-15×106on 1 cm3on pre-saturated environment matrix.

As the matrix can be used, for example, the biomaterial-based polyoxometalate (3-oxybutyrate) - Elastopor. Polyoxometalate - homopolymer synthesized different types of prokaryotic cells. They are the substrate of endogenous respiration and maintain cell viability in non-optimal environmental conditions (Sevastianov V.I., Perova NV, Dovzhik I.A., Titushkin I.A., German E.A., Belomestnykh SM, shyshatskyy E.I., Volova T.G. biomedical properties of polyoxyalkylated - biodegradable bacterial polymers. // Advanced materials, 2001, No. 5. - P.6-55; Sevastianov V.I., Perova NV, Shishatskaya E.I., Kalacheva G.S., Volova T.G., Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood. J. Biomater. Sci. Polymer Edn., 2003. V. 14(10). - P.1029-1042; T. Volova, E. Shishatskaya, Sevastianov, V., Efremov, S., Mogilnaya O., Results of biomedical investigations of PHB and PHB/PHV fibers, Biochem. Eng. J., 2003, No. 3736. - P.1-9).

Elastopor currently approved for medical use. The main component Elastopor is a biodegradable bacterial copolymer of β-oxybutyrate and β-oxovalerate (FOB-with-STAND) with the inclusion of oxovalerate 15÷30 mol.% (Mw=295-360 KDa, crystallinity 50-60%), obtained from the Institute of Biophysics SB RAS, Krasnoyarsk, Russia. Part Elastopor also includes high-molecular hydrophilic plasticizer (AIV), which increases the hydrophilicity and elasticity of the material.

Physico-chemical, mechanical and technological properties Elastopor make this biopolymer is very attractive to develop time frames for hybrid bioisosteric bodies (Sevastianov V.I., Egorov V.A., German E.A., Perova NV, Onishchenko N.A. a Biodegradable biopolymer material Elastopor for cell transplantation. // Advanced materials, 2004, №3, p.35-41; Sevastianov V.I., German E.A., Volkov YEAR, Markovtsev MG three-Dimensional porous matrices for stem-cell-based biodegradable bacterial copolymer Bioplastics". // Advanced materials, 2007. No. 6. P.5-10).

IP is alzhemier the matrices Elastopor had the following dimensions and parameters: diameter of the porous sponge - 1±2 mm; thickness of 1.2±0.5 mm; weight - 6,0-12,0 mg Porosity is not less than 95±2%. The size of the macropores is 300±100 microns.

4). Co-cultivation of allogeneic progenitor bone marrow cells and allogeneic liver cells in vitro.

In the particular case of co-cultivation was performed allogeneic liver cells and progenitor cells from bone marrow according to the standard technique (P.M. Kaufmann, K. Sano, Uyama, S., Breuer C.K., Organ G.M., Schloo B.L., Kluth D., Vacanti J.P. Evaluation of Methods of Hepatotrophic Stimulation in Rat Heterotopic Hepatocyte Transplantation Using Polymers // J. of Pediatric Surgeru. - 1999. - Vol.34. - P.1118-1123; Uyama, S., Kaufmann P.M., Takeda T., J.P. Vacanti Delivery of whole liver equivalent hepatocyte mass using polymer devices and hepatotrophic stimulation // Transplantation. - 1993. - Vol.55. - P.932-935).

This allowed to increase the number of viable liver cells and progenitor cells from the bone marrow, capable of performing specific functions: detoxification and synthetic functional activity of hepatocytes, and created conditions for the differentiation of progenitor cells from the bone marrow into the endothelial cells to form blood vessels and their germination in the matrix.

The matrix with cells were placed in a sterile chamber for incubation in vitro in the growth medium [William's E with the substitution of arginine to ornithine, with the addition of fetal bovine serum, growth factor hepatocytes, epidermal growth factor, β-subunit of the cholera toxin, dexamethasone, ethanolamine, sodium Selenite, glucagon, insulin, insole is about-like growth factor-I, ascorbic acid, linolevoi and linelevel fatty acids] for 2-3 days.

5). Transplantation of media immobilizerturning allogeneic liver cells and progenitor cells in the bone marrow in the body.

Transplantation of matrices with immobilizerturning allogeneic liver cells and allogenic progenitor cells in the bone marrow provide patients with liver failure in the mesentery of the small intestine.

6). The use of anticoagulants and antiagregantov.

After transplantation of matrices with immobilizerturning allogeneic liver cells and allogenic progenitor cells in the bone marrow of patients prescribed anticoagulants in the prevention dose, for example: heparin 5,000 IU every 12 hours for 7-10 days under the control of the blood coagulation system and antiplatelet agents in the prevention dose, for example, trental based 45 mg/m2body surface area every 12 hours for 30 to 90 days.

7). The use of immunosuppressants.

After transplantation of matrices with immobilizerturning allogeneic liver cells and LoginName progenitor cells in the bone marrow of patients prescribed immunosuppressant in the preventive dose, for example: human chorionic gonadotropin 500-5000 ME 1 time per day for 30-90 days.

The proposed transplant for the treatment of hepatic nedostatocno and consists of a three-dimensional biocompatible biodegradable porous matrix and planted him allogeneic progenitor bone marrow cells and allogeneic liver cells.

To prove the possibility of achieving the stated purpose and achievement of the technical result here is the following data.

An example of the proposed method using the proposed transplant experiment.

Modeling of chronic liver insufficiency in animals (rats) were carried out according to recognized and adequate model (Fischer A. Physiology and experimental pathology of the liver. // Afisher. - Budapest, 1961, - 230 S.; Kalashikov IVAN Effect of transplantation of cells of the thymus, bone marrow and spleen on regenerative processes in the diseased liver. // Bul. the experimental. Biol. and med., - 1979, No. 10. - S-480; Kalashikov IVAN General and local changes in the body in experimental liver injury and regeneration. // Abstract. Prof. Diss. - 1982, Kazan. - 41 S.) by introducing a 60% solution of CCl4the first administration of 0.5 ml per 100 g mass, subsequent to 0.3 ml per 100 g body weight. Course introduction 6 weeks with a frequency of 2 introduction in a week.

Modeling of acute liver failure in animals (dogs) was carried out by resection 45-50% of the liver tissue, which is also a recognized and adequate model of acute liver failure (Demetriou, A., Reisher A., Sanchez J. Et al. Transplantation of microcarrier-attached hepatocytes into 90% partially hepatomized rats // Hepatology. - 1988. - Vol.8. - P.1006-1009; Chikoti S. p., Plehanova. Liver failure. //In the book. Liver failure. Irkutsk. - 2002. - 260 S.; G.K. Michalopoulos Liver regeneration: Molecular mechanisms of growth control // FASEB J. - 1990. - Vol.104. - P.176; Bowling WM, Kennedy SC, Cai SR, Duncan JR, Gao C, Flye MW Portal branch occlusion safely facilitates in vivo retroviral vector transduction of rat liver. // Hum. Gen. Ther/ - 1996. - vol.7(17). - p.2113-2121).

1). Carried out according to traditional methods, the selection of allogeneic progenitor cells from the bone marrow.

Received cellular aspirate of bone marrow canal of the tibia bone (rats) or humerus (dogs) by lavage of a phosphate buffer solution containing 50 u/ml heparin and 0.25 mg/l of gentamicin using 18G needle mounted on the syringe. Cell suspension was centrifuged, the precipitate cells resuspendable in lytic solution at room temperature for 3 min and centrifuged for 3-5 min at 1500 rpm Hamalsarany supernatant was completely removed by aspiration, and the cell precipitate containing progenitor cells in the bone marrow resuspendable in the growth environment. The interphase with mononuclear cells were collected from the surface of erythroid sediment and resuspendable in lytic solution, at a ratio of 1:4, for 5-8 min and centrifuged 5 min at 1500 rpm at room temperature. Hamalsarany supernatant was completely removed. Achieved complete lysis of red blood cells, for which the procedure of Lisino the project conducted two or three times with the subsequent laundering of the cells by centrifugation. Cellular precipitate free of erythroid and platelet forms, in the amount of 60-150×106cells were combined with sediment cells derived from plasma, and then resuspendable in the growth environment to stimulate cell growth.

2). Selection allogenic liver cells was carried out according to traditional methods.

The selection of allogenic liver cells from resected area of the liver (4×4×2 cm in dogs and 1×1×0.5 cm in rats) was performed by 3 times washing with blood and grinding it to a cold (t=4°C), with 3 times washing the resulting suspension buffer solution without calcium within 7 minutes. After this, small pieces of liver were preincubator 3 times with a solution of collagenase for 6-8 minutes, followed by replacing the enzyme solution using centrifugation at 500 rpm for 1 minute at t=37°C. the resulting material was transferred to a sieve with meshes of 200 μm and was filtered washing with culture medium William's E with 10% fetal bovine serum, after which the cell suspension was centrifuged at 500 rpm at 4°C for 1 minute. The supernatant was removed, the residue resuspendable in the same fresh medium and again centrifuged. The procedure was repeated 3 times. Cell viability, just in the range from 83 to 95% was estimated by the method of dyeing Trifanova blue. The number of cells ranged ot,0×10 8to 4.0×108hepatocytes on 15 g of liver tissue. The cell suspension contained: hepatocytes ~95÷98%, and ~5÷2% neprekonatelnych liver cells, which were identified by light microscopy. Division prehistoric and neprekonatelnych cells was not performed. A suspension of liver cells was concentrated to 1-2 ml of physiologic R-RA.

3). Landing (immobilization) of allogenic liver cells and progenitor cells from the bone marrow to the media was also carried out according to traditional methods.

Allogene liver cells and progenitor cells in the bone marrow resuspendable in the growth medium at a concentration of 2.0 to 4.0×106liver cells/ml and 2,0-4,0×106progenitor bone marrow cells/ml Total cell suspension was applied to the matrix at a concentration of 2×106-15×106on 1 cm3on the pre-saturated environment matrix.

4). In the particular case of co-cultivation was performed allogeneic liver cells and progenitor cells from bone marrow according to the usual method.

This allowed to increase the number of viable liver cells and progenitor cells from the bone marrow, capable of performing specific functions: detoxification and synthetic functional activity of hepatocytes, and to create conditions for the differentiation of progenitor cells from the bone marrow in the endothelial cells for fo the formation of the vessels and their germination in the matrix.

The matrix with cells were placed in a sterile chamber for incubation in vitro in the growth medium for 2-3 days.

As the matrix was used, for example, the biomaterial-based polyoxometalate (3-oxybutyrate) Elastopor. Elastopor has a molecular weight of 295-360 KDa synthesized by the Institute of Biophysics SB RAS (Krasnoyarsk), and is currently approved for medical use.

Used matrices Elastopor had the following dimensions and parameters: diameter of the porous sponge - 10±2 mm; thickness of 1.2±0.5 mm; weight - 6,0-12,0 mg Porosity is not less than 95±2%. The size of the macropores is 300±100 microns.

5). Transplantation of matrices with immobilizerturning allogeneic liver cells and progenitor cells in the bone marrow in the body of animals (dogs) was carried out for 3-4 days after modeling liver failure by resection of 50% of the liver tissue.

Before transplantation, animals (dogs) were narcoticyou: a mixture of drugs was carried out by intramuscular injection of a mixture of products: Calypsol (5% of 10 mg/kg Droperidol (1.5 mg/kg), Atropine (0.1% to 0.02 mg/kg). Diphenhydramine 1% to 0.5 ml and then, in the operating room was performed anaesthesia induction by intravenous injection of 1% Propofol at the rate of 7 mg/kg. the Operation was performed under endotracheal anesthesia. Maintenance of anesthesia was performed with 1% Propofol 0.5 mg/kg/h and Kalipsola 2 mg/kg In rats done by ulali anesthetic allowance calculated on 100 g of body weight were injected intramuscularly: Calypsol 10% - 2 ml, Xylazine 2% - 1 ml Atropine - 0,2 ml

Total cellular material containing allogeneic liver cells and bone marrow at a concentration of 2×106-15×106on cm3that is immobilized on the matrix Elastopor, transplanted in the mesentery of the small intestine of animals.

The abdominal cavity is sutured in layers tightly. For the prevention of infectious complications intraoperatively animals were intravenously administered drove Ofloxacin 40 ml; Metronidazole - 30 ml; Ditilin 5% to 20 mg/kg/h of the monitored indicators of biochemistry, coagulation, blood count at 1, 3, 9, 14, 18 days after the operation.

6). The use of anticoagulants and antiagregantov.

In the particular case to prevent cell infiltration of the graft by the red blood cells immediately after transplantation matrices with immobilizerturning by autologous liver cells and autologous progenitor cells in the bone marrow daily was prescribed anticoagulants in the prevention dose, for example: heparin 2500 ME every 12 hours for 7-10 days under the control of the blood coagulation system and antiplatelet agents profilakticheski dose, for example: trental based 45 mg/m2body surface area every 12 hours for 30 to 90 days.

7). The use of immunosuppressants.

In the particular case for the prevention of rejection immediately after transplantation matrices with them is abilityone allogeneic liver cells and LoginName progenitor cells in the bone marrow every day appointed by immunosupressant in the preventive dose, for example: human chorionic gonadotropin 500-5000 ME 1 time per day for 30-90 days.

In total we performed 20 experiments, and the correction of acute liver failure under the proposed method was carried out 3 experiments and chronic liver failure - in 17 experiments.

Figure 1 presents the dynamics of synthetic liver function (total protein) in acute liver failure. Studies have shown that after resection of 50% of the liver is minimal synthetic function of the liver was observed during the first 3 days after resection. Compared to outcome indicators total protein decreased 1.6 times, which indicates a significant functional impairment of liver cells. Then a gradual return of these indicators to the original to 18 days after modeling liver failure. In the experiment after transplantation matrix with planted at him allogeneic progenitor cells in the bone marrow and allogeneic liver cells under the proposed method, the dynamics of the synthetic function of the liver was similar to the control group. However, since transplantation (3 days after modeling liver failure) allogeneic liver cells and progenitor cells from the bone marrow, planted on the matrix, there faster Voss is the resolution to baseline liver synthetic function. Moreover, it should be noted that in the control group in the study period (18 days after the operation) this indicator has not reached the source. This only happened to 28-32 days after the operation.

Figure 2 presents the dynamics of the synthetic function of the liver (cholesterol) in acute liver failure. Studies have shown that after resection of 50% of the liver is minimal synthetic function of the liver was observed during the first 3 days after resection. Compared to outcome indicators cholesterol decreased by 1.64 times, which indicates a significant functional impairment of liver cells. Then a gradual return of these indicators to the source in the study group to 18 days after modeling liver failure, and in the control group in the study period (18 days after the operation) this indicator (cholesterol) and has not reached the source. This only happened to 28-32 days after the operation.

From the presented data it follows that until allogenic liver cells and progenitor cells from the bone marrow, planted on the matrix, the dynamics of indicators characterizing the synthetic function of the liver was similar in both groups (control and experiment), however, in the study group (experiment) the restoration of the synthetic function of the liver (on the be the ku and cholesterol) was faster and these figures were closer to the source.

Figure 3 presents the group viable allogenic hepatocytes (1) 90 days after their transplantation into the body of an experimental animal under the proposed method.

Color: hematoxylin - eosin. The magnification ×400.

Figure 4 presents these histological examination after 90 days after transplantation of allogenic liver cells and progenitor cells from the bone marrow, planted on the matrix. In histological preparations of the animals who did not receive anticoagulants and antiplatelet agents, revealed severe inflammatory reaction (2), fibrotic changes (3), proliferation of hepatocytes (4).

Color: hematoxylin - eosin. The magnification ×200.

Figure 5 in histological preparations of the animals who did not receive anticoagulants and antiplatelet agents, and immunosuppressant after 180 days after transplantation of allogenic liver cells and progenitor cells from the bone marrow, planted on the matrix revealed severe inflammation, lymphoid reaction (5).

Color: hematoxylin - eosin. The magnification ×200.

Figure 6 animals that received anticoagulants and antiplatelet agents and immunosuppressant after 180 days after transplantation, revealed significantly lower cell response. In the preparation of meet up groups of viable hepatocytes (6).

Color: heme is oxilan - eosin. The magnification ×400.

7 after 180 days on the proposed method in the product revealed the newly formed vessel (7).

Coloring PAS. The magnification ×200.

In all cases, was achieved the desired result, and it was received and adequately compensated liver failure. The method can improve the results of treatment of liver failure by providing isolated allogeneic hepatocytes detoxification and bioregulatory functions, extension of the terms of their survival by activation of proliferation through joint intracorporeal injection of allogeneic progenitor cells in the bone marrow, create a frame for placement in the space of associations formed cells, creating conditions for germination in this framework vessels, diffusion of nutrients, oxygen and factors of tissue differentiation of the blood vessels of the mesentery, sprouted in the frame, to the immobilized liver cells and bone marrow cells; provides for prevention of complications associated with the use of graft cells. In addition, prolonged use of viable allogenic liver cells and progenitor cells from the bone marrow allows long-term to maintain homeostasis.

Thus, the above data clearly indicate that the correction of liver failure is much faster and more appropriately in the application of the proposed method of correction of liver failure. In addition we have proved long-term survival of liver cells and the appearance of newly formed vessels in the device after 90 and 180 days after transplantation animal matrix with allogeneic liver cells and allogenic progenitor cells in the bone marrow.

Given the above, the extrapolation of experimental results to the clinic legitimate, the proposed method and the graft, allowing the prolongation of life of isolated hepatocytes can be used for the correction of liver failure.

1. A method for the treatment of liver failure, including the use of isolated liver cells, characterized in that the taking of allogeneic progenitor bone marrow cells and allogeneic liver cells from the donor, perform landing freshly isolated allogeneic liver cells and allogeneic progenitor bone marrow cells on three-dimensional biocompatible biodegradable porous matrix with a pore size of 2-500 μm and a total porosity of 50-97%, providing a total concentration of liver cells and progenitor cells from the bone marrow 2·106-15·106cells on 1 cm3matrix and the ratio of the progenitor cells from the bone marrow to the liver cells from 1:1 to 1:4, and the total amount of the matrix must be not less than 0.05 cm3and it's lowest line is hydrated size should be not less than 0.2 mm, and transplanted matrix with the cells of the liver and bone marrow in the mesentery of the small intestine.

2. The method according to claim 1, characterized in that after landing allogeneic liver cells and progenitor cells from the bone marrow to the matrix prior to transplantation produce their co-cultivation.

3. The method according to claim 1, characterized in that immediately after transplantation of allogenic liver cells and progenitor cells from the bone marrow to the matrix prescribe anticoagulants and antiplatelet agents in the prevention dose.

4. The method according to claim 1, characterized in that immediately after transplantation of allogenic liver cells and progenitor cells from the bone marrow to the matrix prescribe immunosuppressants in the preventive dose.

5. The method according to claim 2, characterized in that the co-cultivation of allogenic liver cells and progenitor cells from the bone marrow is carried out in 2-3 days.

6. The transplant for the treatment of liver failure, including a three-dimensional biocompatible biodegradable porous matrix having a total volume of not less than 0.05 cm3and the smallest linear dimension of at least 0.2 mm, a pore size of 2-500 μm, the total porosity 50-97%, and planted him allogeneic progenitor cells in the bone marrow and allogeneic liver cells, and the concentration of liver cells and bone marrow 2·106-15·106cells on 1 cm3the matrix is the ratio of bone marrow cells to liver cells from 1:1 to 1:4.



 

Same patents:

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and can be applied for treatment of liver failure. Transplant includes heterogenic biocompatible biodegradable gel, which has total volume not less than 0.1 ml and the smallest linear size not less than 0.2 mm, pore size 30-500 mcm, total porosity 50-98%, implanted on it autologous progenitor cells of bone marrow after their cultivation in vitro and cultivated autologous liver cells, concentration of liver and bone marrow cells being 2×106-15×106 cells per of 1 cm3 of heterogeneous gel and ratio of bone marrow cells to liver cells being from 1:1 to 1:4. In method of treating liver failure transplant is placed into parenchyma of liver and/or mesentery of small intestine.

EFFECT: group of inventions makes it possible to increase term of cell survival, activate their proliferation.

4 cl, 6 dwg

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and can be applied for treatment of liver failure. Transplant for treatment of liver failure includes heterogenic biocompatible biodegradable gel, which has total volume not less than 0.1 ml and the smallest linear size not less than 0.2 mm, pore size 30-500 mcm, total porosity 50-98%, implanted on it autologous progenitor cells of bone marrow after their cultivation in vitro and cultivated autologous liver cells, concentration of liver and bone marrow cells being 2×106-15×106 cells per 1 cm3 of heterogeneous gel and ratio of bone marrow cells to liver cells being from 1:1 to 1:4. In realisation of method of treating liver failure transplant is placed into parenchyma of liver and/or mesentery of small intestine.

EFFECT: group of inventions makes it possible to increase term of cell survival, activate their proliferation.

4 cl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to chemical-pharmaceutical industry, namely to application of an extract of an elevated part of scabiose centaury (Centaurea scabiosa L.) as an antioxidant hepatoprotector. A method of preparing the antioxidant hepatoprotector consists in grinding the elevated part of scabiose centaury (Centaurea scabiosa L.) to particle size 2-4 mm followed by three-fold extraction in 70% ethanol at temperature 75-80°C for 1-1.5 hours in the raw material to extractant relation 1:15-1:20, evaporation to dryness of the prepared extract.

EFFECT: application of the extract of the elevated part of scabiose centaury (Centaurea scabiosa L) allows extending the range of herbal antioxidant hepatoprotectors, promotes normalising the antioxidant activity in hepatic tissue, regression of the main biochemical values in hepatitis.

2 cl, 20 ex, 9 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely, to efferent therapy, and can be applied in treatment of patients with hepatic failure after performed cardio-surgical operation. For this purpose realised is veno-venous hemofiltration with application of standard bicarbonate solution. If clinical and laboratory signs of hepatic failure increase following sessions of hemofiltration are carried out during 5-8 hours with application of dialytic solution based on standard bicarbonate solution with 2% content of albumen. After session of hemofiltration with such solution, laboratory control of basic functional hepatic indices is performed. In case of necessity additional sessions with application of dialytic solution which contains 2% albumen are carried out.

EFFECT: method makes it possible to increase efficiency of small-flow veno-venous hemofiltration due to increase of clearance of endotoxins, associated with blood plasma proteins.

2 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to abdominal surgery, hepatology and methods of detoxication, and can be applied for treatment of liver abscess of different etiology. For this purpose after sanitisation of purulent nidus cavity of liver abscess is processed with 0.25% solution of derinate, then blood sampling is carried out in quantity 150-200 ml of blood, after centrifugation of which erythrocytes are returned to patient, and leucocytes are extra-corporeally processed with 0.5% solution of glutoxim in dose 1 ml and diluted in 50-100 ml of 0.9% solution of NaCl with following intravenous drop introduction, and from the following day into cavity introduced is 0.25% solution of derinate in dose 1.5 ml one time per day during 5 days.

EFFECT: method makes it possible to reduce treatment duration and reduce number of complications due to improvement of indices of cellular link activity, connected with increase of quantitative indices of T-helpers and T-suppressors at the background of increase of phagocytic activity of neutrophils, as well as reduction of erythrodieresis processes due to normalisation of APFC content and humoral link of immunity.

2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula I, which are HSP90 (heat-shock proteins) inhibitors and can be used to prepare a medicinal agent for treating tumorous diseases affected by HSP90 inhibition. In formula I R1 denotes Hal, H, OA or A, R2, R3 each independently denotes -O-(X)s-Q, -NHCO-(X)s-Q, -CONH-(X)s-Q, -NH(CO)NH-(X)s-Q, -NH(CO)O-(X)s-Q, -NHSOr(X)s-Q, NHCOA, Hal, Het or H, where, if R2=H, then R3≠H, or if R3=H, then R2≠H, R4 denotes H, R5 denotes H, Hal, A, OA, (CH2)nCOOH, (CH2)nCOOA, O(CH2)oCONH2, NHCOOA, NHCO(CH2)nNH2, NHCONHA or O(CH2)oHet1, A denotes a straight or branched alkyl containing 1-10 carbon atoms, in which 1-5 hydrogen atoms may be substituted with F, Cl and/or Br, X denotes a straight or branched C1-C10 alkylene which is unsubstituted or substituted once, twice or thrice by A, O A, OH, Hal, CN, COOH, COOA, CONH2, NH2, NHCOA, NHCOOA, Q denotes H, Ar or Het, Ar denotes phenyl which is unsubstituted or substituted once, twice or thrice with A, OA, OH, NO2, Hal, CN, (CH2)nCOOH, (CH2)nCOOA and/or tetrazole, Het denotes a cyclic saturated or aromatic 5-6-member heterocycle containing 1-2 N and/or O atoms, optionally condensed with a benzene ring which may be substituted once, twice or thrice with A, OA, OH and/or =O (carbonyl oxygen), Het1 denotes a monocyclic saturated, unsaturated or aromatic heterocycle containing 1-2 N and/or O atoms, which may be mono- or disubstituted with A, OA, OH, Hal and/or =O (carbonyl oxygen), Hal denotes F, Cl, Br or I, n equals , 1, 2, 3 or 4, o equals 1, 2 or 3, s equals 0, 1 or 2.

EFFECT: high efficiency of using said derivatives.

4 cl, 4 dwg, 1 tbl, 29 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to drugs, and concerns a therapeutic agent for treating hepatitis containing 2-amino-2-[4-3-benzyloxyphenylthio)-2-chlorophenyl]ethyl-1,3-propanediole or its pharmaceutically acceptable salt, or hydrate as an active ingredient. Also, disclosed is a method of treating hepatitis by introducing to a patient an effective amount of the therapeutic agent 2-amino-2-[4-3-benzyloxyphenylthio)-2-chlorophenyl]ethyl-1,3-propanediole or its pharmaceutically acceptable salt, or hydrate as an active ingredient.

EFFECT: therapeutic agent shows a stronger effect in treating hepatitis as compared to the other analogues.

4 cl, 3 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine and is intended for treatment of gastroesophageal reflux disease (GERD) in patients with chronic opisthorchosis. PH-monitoring of upper parts of GIT and dehelmintisation are carried out. Type of reflux is determined. Preparations of ursodeoxycholic acid (UDCA) are administered. In case of mixed type of GER with prevalence of acid component dose is 250 mg/day for 2 weeks. In case of mixed type of GER with prevalence of alkali component dose is 500 mg/day before going to bed for 4 weeks. In isolated alkali version dose is 10 mg/kg in two intakes: morning and evening. In case of tgenpH>7 from 16.54 to 27.6%; NpH>7 from 27 to 31, and GER tgenpH>7 from 27.6 to 48.8%; NpH>7 from 31 to 35 - for 4 weeks. In case of tgenpH>7 from 16.54 to 27.6%; NpH>7 from 27 to 31, and GER tgenpH>7 from 27.6 to 48.8%; NpH>7 from 31 to 35 and in case of GER tgenpH>7 from 48.8 to 74.4%, NpH>7 from 36 to 42 - for 8 weeks. In case of tgenpH>7 from 48.8 to 74.4%; NpH>7 from 36 to 42, and GER tgenpH>7 from 16.54 to 74.4%; NpH>7 from 27 to 42 -for 12 weeks.

EFFECT: method makes it possible to reduce motor-tonic disturbances of biliary tract, reduce number of recurrences, enhance treatment efficiency.

4 cl, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of pharmacy, in particular to phytotherapy, and can be applied for treatment of drug hepatitis. Medication represents herbal mixture, including 29 herbs: herbal part of woodland strawberry, herbal part of common dandelion, inflorescences of pineapple weed, leaves of stinging nettle, inflorescences of tansy, inflorescences of pot marigold, fruits of common juniper, peppermint leaves, herbal part of St John's wort. Medication additionally contains: lime tree flowers, herbal part of heartsease, herbal part of Centaurium, flax seeds, dill seeds, burdock root, herbal part of common chicory, herbal part of common knotgrass, collective fruits of common hop, herbal part of cornflower, leaves of cowberries, leaves of Arctostaphylus, corn stigma, herbal part of immortelle, root and rhizome of Acorus, herbal part of Menyanthes, garden angelica root, herbal part of yellow sweet clover, Inula root, herbal part of common horsetail.

EFFECT: mixture reduces terms of hepatitis treatment, as well as improves clinico-laboratory indices due to normalisation of liver function tests and functional state of liver.

4 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine. Mixture consists of medicinal plants: inflorescences of pot marigold, peppermint leaves, Baikal skullcap root, black leaves and root of badan, shoots of shrubby cinquefoil, with the following component ratio, wt fr.: inflorescences of pot marigold 3; peppermint leaves 1; Baikal skullcap root 2; rhizome and black leaves of badan - 1+1; shoots of shrubby cinquefoil 2. Clinical treatment with claimed mixture has resulted in positive effect in case of alcoholic hepatitis in 150 patients.

EFFECT: creation of mixture for treatment and prevention of alcoholic hepatitis.

10 tbl

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and can be applied for treatment of liver failure. Transplant includes heterogenic biocompatible biodegradable gel, which has total volume not less than 0.1 ml and the smallest linear size not less than 0.2 mm, pore size 30-500 mcm, total porosity 50-98%, implanted on it autologous progenitor cells of bone marrow after their cultivation in vitro and cultivated autologous liver cells, concentration of liver and bone marrow cells being 2×106-15×106 cells per of 1 cm3 of heterogeneous gel and ratio of bone marrow cells to liver cells being from 1:1 to 1:4. In method of treating liver failure transplant is placed into parenchyma of liver and/or mesentery of small intestine.

EFFECT: group of inventions makes it possible to increase term of cell survival, activate their proliferation.

4 cl, 6 dwg

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and can be applied for treatment of liver failure. Transplant for treatment of liver failure includes heterogenic biocompatible biodegradable gel, which has total volume not less than 0.1 ml and the smallest linear size not less than 0.2 mm, pore size 30-500 mcm, total porosity 50-98%, implanted on it autologous progenitor cells of bone marrow after their cultivation in vitro and cultivated autologous liver cells, concentration of liver and bone marrow cells being 2×106-15×106 cells per 1 cm3 of heterogeneous gel and ratio of bone marrow cells to liver cells being from 1:1 to 1:4. In realisation of method of treating liver failure transplant is placed into parenchyma of liver and/or mesentery of small intestine.

EFFECT: group of inventions makes it possible to increase term of cell survival, activate their proliferation.

4 cl

FIELD: medicine, veterinary science.

SUBSTANCE: method involves injections to animals of hepatic tissues hydrolysate and mineral salts of isotonic concentration in effective doses.

EFFECT: method allows reducing disease incidence, improving safety of livestock, increasing effectiveness and reducing treatment time.

4 cl, 5 tbl, 5 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary science, particularly to agents and methods of treating keratoconjunctivitis in cattle. A preparation for treatment and prevention of infectious keratoconjunctivitis in cattle contains an aqueous solution of Sulfur, Echinacea purpurea, Hepar sulphur, Belladonna albus, Apis melifelica and tissue nosode in the following relation, wt %: Belladonna albus C6 - 10, Sulfur C6 - 20, Echinacea purpurea C6 - 10, Apis melifelica C6 - 30, Hepar sulphur C6 - 15, tissue nosode D6 - 15. The method for treatment and prevention of infectious keratoconjunctivitis in cattle involves intramuscular introduction of said preparation to calves, cows and heifers once a day every 3-5 days, and to calves to 80 kg of live weight the preparation is introduced in a dose 1-2 ml/10-15 kg of live weight, and to cows and heifers in a dose 1-2 ml/100 kg of live weight.

EFFECT: preparation and method exhibit high therapeutic efficiency in comparison with analogues.

4 cl, 4 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and namely to hepatology and biotechnology, and deals with treatment of chronic diffusion liver diseases as well as treatment of liver cirrhosis and portal hypertension by means of the biotransplant (BT) obtained for that purpose. BT contains mixed cell pool in which 40% comprise stem cells, including stromal mesenchymal and hepatocyte bipotent stem cells, and the rest 60% - progenitor cells in the various phase of differentiation, including new immature hepatoblasts and progenitor cells of erythroid row, hemopoietic cells. Mixed BT cell pool is characterised with the expression of the following surface markers: CD13, CD29, CD44, CD90, CD117 (c-Kit) and absence of CD34 expression. For the purpose of treatment there injected is the above BT in the form of suspension in physiological solution in quantity of 2-4 mln cells per 1 kg of the patient's weight. At liver cirrhosis and portal hypertension, BT is injected in the form of suspension in physiological solution at total number of BT cells of 350 to 500 mln.

EFFECT: BT injection given above is an independent treatment method of liver diseases, which provides stable decrease of the process activity, decrease of degenerative dystrophic changes, and recurrence-free period of 12 months long.

3 cl, 3 ex, 9 tbl, 12 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns biopharmaceutics and PEG conjugates of natural or recombinant urate oxidase (uricase). Uricase is bound covalently to poly(ethylene glycol) or poly(ethylene oxide) (both denoted as PEG), with average of 2 to 10 PEG threads are conjugated with each uricase sub-unit, and average molecular weight of PEG is approximately between 5 kDa and 100 kDa.

EFFECT: obtaining almost non-immunogenic PEG uricase conjugates preserving at least 75% of uricolytic activity of non-modified enzyme.

44 cl, 12 ex, 17 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, in particular to hepatic cells production, and may be used in medical science. From the whole liver or resected part thereof, a cell population enriched with living cells of human liver, including hepatic stem cells/precursor cells, is obtained. Cell population contains functional hepatocytes and biliary cells expressing cytokeratin 19 (CK19), but not expressing albumin, as well as hepatic stem cells/precursor cells 9 to 13 mcm in diameter and expressing EP-CAM, CD 133 markers. Resulting cell population is used for hepatotherapy.

EFFECT: production of living population of hepatic cells sufficiently efficient for regeneration.

60 cl, 16 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, to gynecology in particular. It is designated for optimization of venter metriosis patients treatments in clinical trial. For this purpose patients of the reproductive age intake "Skvaakan" in addition to a hormonal medicine. The intake is implemented intramuscularly, 2.0 ml per day during 75 days with septan interruption after each 30 days. This method contributes to reduction of treatment terms, disease backsets, reactivation of reproductive function among the patients of this category.

EFFECT: reduction of treatment terms, disease backsets, reactivation of reproductive function.

3 cl, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to human stem hepatocytes. Offered composition includes primitive human stem hepatocytes expressing Ep-CAM, AC 133 and albumin, and culture medium. These primitive human stem hepatocytes are precursors of proximal stem hepatocytes that are precursors of hepatocytes or precursors of bile ducts. Offered method of human cells liver-tissue precursors release includes identification of cells expressing Ep-CAM, AC 133 and albumin. Offered method of human hepatocytes precursors released by method under cl.15 or 16, cells precursors are primitive human stem hepatocytes expressing Ep-CAM, AC 133 and albumin. Offered method of human liver-tissue hepatocytes precursors release includes identification of cells expressing Ep-CAM. Offered method implies primitive human stem hepatocytes release expressing Ep-CAM, AC 133 and albumin. Offered primitive human stem hepatocytes expressing Ep-CAM, AC 133 and albumin released by method under cl. 26. Offered method implies proximal human stem hepatocytes release. Offered method provides treatment of liver dysfunction and diseases including introduction of primitive human stem hepatocytes in effective amount. Released primitive human stem hepatocytes are offered.

EFFECT: invention enables to apply produced compositions for cell therapy and bioartificial organs.

59 cl, 11 ex, 4 tbl, 20 dwg

FIELD: medicine.

SUBSTANCE: it has been suggested the method for introducing a preparation in efficient dosage being either a substance or preparation prepared according to homeopathic technique or biologically active additive that contains a substance isolated out of hepatic cells being slightly hydrophobic, water-soluble, negatively charged at alkaline pH, at molecular weight ranged 500-15000 Da that enables to decrease tissue-specifically the ratio of phosphorylated adenosine diphosphate against the quantity of atomic oxygen spent by highly energized mitochondria in the course of oxidizing phosphorylation. The suggested method and preparation enable to treat efficiently astheno-depressive state and, also, increases the efficiency of complex therapy of pulmonary and intestinal diseases.

EFFECT: higher efficiency of therapy.

16 cl, 21 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and can be applied for treatment of liver failure. Transplant includes heterogenic biocompatible biodegradable gel, which has total volume not less than 0.1 ml and the smallest linear size not less than 0.2 mm, pore size 30-500 mcm, total porosity 50-98%, implanted on it autologous progenitor cells of bone marrow after their cultivation in vitro and cultivated autologous liver cells, concentration of liver and bone marrow cells being 2×106-15×106 cells per of 1 cm3 of heterogeneous gel and ratio of bone marrow cells to liver cells being from 1:1 to 1:4. In method of treating liver failure transplant is placed into parenchyma of liver and/or mesentery of small intestine.

EFFECT: group of inventions makes it possible to increase term of cell survival, activate their proliferation.

4 cl, 6 dwg

Up!