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 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.

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The invention relates to medicine, transplantation, cell transplantation, 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 method of treatment pictocin the th 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., 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 the Honey. - 1997. No. 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).

Also known SPO is about the treatment of liver failure, assuming using a new in vitro system filled with collagen gel with hepatocytes (Naka, S., Takeshita, K., Yamamoto T. Bioartificial liver support system using porcine hepatocytes entrapped in a three-dimensional hollow fiber module with collagen gel: an evaluation in the swine acute liver failure model // Artif. Organs. -1999. - V. 23. - P.822-828).

Also known is a method of treatment and correction of liver failure by intramuscular implantation of a pool of isolated hepatocytes in micro - or microcapsule (Hunkeler D., Cherrington, A., Prokop, A., Rajotte R. Bioartificial Organs III. Tissue Sourcing, Immunoisolation, and Clinical Trials. // Annals of the New York. Academy of Sciences. - N.Y., 2001. - V.944).

As a prototype we have chosen a method of treatment of liver failure, which were 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;

- neobhodimosti use of 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, since with increasing time of cultivation 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".

- inability to create three-dimensional structure, similar to the architectonics with the tissue of the liver.

A device for the treatment of liver is insufficient the particular method of in vitro connection of isolated hepatocytes (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). 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 heterotropically transplanted liver grafts // Transplantation. - 2001 - Vol.7. - P.1-7).

The disadvantages of the known devices, including prototypes, based on biomodule 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 is 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, oxygen and factors of tissue differentiation of the blood vessels of the liver and/or 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 thrombosis 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 heterogeneous gel culture conditions for Strait is perezii cells and the formation of a "tissue-like" structure;

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

- the use of autologous cells, which reduce the degree of activation of the immune system, allow the 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.

This method contributes to a more rapid integration of the device in 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 and antiplatelet agents immediately after transplantation inhibits the cellular response to the transplant as foreign body and prevents the formation of thrombi at the site of transplantation device.

The essence of the invention is as follows.

For the treatment of liver failure use of isolated liver cells. Moreover, pre-exercise C the boron autologous progenitor cells from the bone marrow. Carry out culturing them in vitro. Then taking autologous liver cells. Perform landing freshly isolated liver cells and progenitor cells from the bone marrow on heterogeneous biocompatible biodegradable gel. The heterogeneous pore size of the gel varies from 30 to 500 μm. The total porosity of heterogeneous gel is 50-98%. The concentration of total liver cells and bone marrow is 2×106-15×106cells on 1 cm3heterogeneous gel. The ratio of bone marrow cells to liver cells from 1:1 to 1:4. And the total amount of heterogeneous gel not less than 0.1 ml. Least amount of gel should be not less than 0.2 mm Transplantation gel liver cells and bone marrow produce in the damaged parenchyma of the liver and/or in the mesentery of the small intestine.

Cultivation of bone marrow cells is carried out for, e.g., 7 days.

In the particular case before transplantation carry out the co-cultivation of autologous 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, for example, trental based 45 mg/m2the surface of the body of each is haunted 12 hours within 30-90 days.

The proposed transplant for the treatment of liver failure include heterogeneous biocompatible biodegradable gel and planted on him autologous bone marrow cells after in vitro cultivation and freshly isolated autologous liver cells. The total volume of the gel is not less than 0.1 ml, and the smallest linear dimension is not less than 0.2 mm pore Size of the gel 30-500 μm, the total porosity 50-98%. Concentration of liver cells and bone marrow 2×106-15×106cells on 1 cm3gel 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. Transplantation is not allogeneic, and autologous cells 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 gel, stimulates their proliferation, which is supported by diffusion in the gel of nutrients, oxygen and factors of tissue differentiation of blood flowing to the gel of the vessels of the liver or mesenteric;

2. To create a frame-based heterogeneous biocompatible the x biodegradable gels, simulating 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 of the graft and its supporting cells in a viable state through the use of anticoagulants and antiplatelet agents.

The method is as follows, and it includes several successive steps:

1). The allocation of autologous progenitor cells in the bone marrow.

The allocation of progenitor cells from the bone marrow carried out according to traditional methods (Shumakov V.I., Onishchenko N.A., 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). For 4-7 days before the main surgical intervention under local anesthesia dotted line in the iliac bone of the patient 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. Suspension glue is OK centrifuged 5 min at 1500 rpm, sediment 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 completely removed by suction. 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). Cultivation of autologous progenitor cells from bone marrow in vitro.

Culturing progenitor cells in the bone marrow carried out according to traditional methods (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.77-100). Partially purified progenitor cells in the bone marrow were seeded for cultivation in Petri dishes (d=60 mm, the number of 1.5-2.0 million cells/ml were Cultured at 37°C in CO2the incubator atmosphere of 5% CO2and 95% humidity for 7 days with a single change of environments is on the third day. A week later, the culture of bone marrow cells contained up to 10% adherent to plastic spread fibroblast-like and macrophage cells and up to 90% of free-floating in suspension, not rounded adherent cells (hematopoietic cells). Neprecejusies to the plasticity of progenitor cells in the bone marrow was collected and then used for immobilization on heterogeneous gel with liver cells.

3). The allocation of autologous liver cells.

Produce resection of 2-4×2-4×1-2 cm of the liver tissue in patients with liver failure to obtain liver cells. The allocation of autologous cells of the liver are carried out by traditional methods (O. Seglen Preparation of isolated rat liver cells. // Methods. Cell. Biol. 1976. - vol.13. - p.29-83; 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 (l). - 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):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):941-947).

The allocation of autologous liver cells produce of the resected portion of the liver by 3-fold 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 distilled the odes, 8.3 g of NaCl, 0.5 g KCl, 2.38 g HEPES, pH of 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 CaCl, 2.38 g HEPES, 7.5 mg trypsin inhibitor and 500 mg of collagenase Type IV-S (>125 CDU/mg), pH 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 nutrient environment 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 4.0×108hepatocytes 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 physiologic R-RA.

4). Landing (immobilization) autologous liver cells and autologin the bone marrow cells in a heterogeneous gel.

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. Mater. Res. - 1997. - Vol.5. - P.413-420).

Autologous liver cells and autologous 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-I, 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 at a concentration of 2×106-15×106on 1 cm3gently mixed with a heterogeneous gel in a ratio of 1:1, until a uniform consistency was collected in a syringe and kept until the introduction in the liver and/or mesentery at 4°C for not more than 5 hours.

As a heterogeneous gel can be used, for example, a biomaterial, "Spherogel", made on the basis of collagen farm animals (70%) and collagen extract (30%)with the appearance of a thick jelly-like substance.

Heterogeneous gels are used as the element is provided in vitro systems, to encapsulate different types of cells (β-cells, hepatocytes, genetically altered cells, stem cells and growth factors and other (M.S. Shoichet, Li, R.H., White, M.L. et al. Stability of hydrogels used in cell encapsulation: An in vitro comparison of alginate and agarose // Biotechnol. Bioeng. - 1996. - V.50. - P.374-381; Naka, S., Takeshita K., Yamamoto T. Bioartificial liver support system using porcine hepatocytes entrapped in a three-dimensional hollow fiber module with collagen gel: an evaluation in the swine acute liver failure model // Artif. Organs. - 1999. - V.23 supported. - P.822-828; Tan, W., Desai, M. S., T. A. Desai Microfluidic patterning of cells in increasing interest among matrix biopolymers: effect of channel size, cell type, and matrix composition on pattern integrity // Tissue Engineering. - 2003. - V.9. No. 2. - P.255-268; Hedberg LE, Kroese-Deutman H.C, Shih S. Kaliev et al. Effect of varied release kinetics of the osteogenic thrombin peptide TP508 from biodegradable polymeric scaffolds on bone formation in vivo II J. Biomed. Mater. Res. - V.72A. No. 4. - 2005. - P.343-353; Allemann F., S. Mizuno et al. Effect of hyaluronan on engeneered articular cartilage of increasing interest among matrix gene expression in 3-dimensional collagen scaffolds // J. Biomed. Mater. Res. - 2001. - V. 55. - P.13-19; Houweling D.A., Lankhorst AJ, Gispen W.H. Collagen containing neurotrophin-3 (NT-3) attracts regrowing injured corticospinal axons in the adult rat spinal cord and promotes partial functional recovery // Exp. Neurol. - 1998. - V.153. No. 1. - P.49-59; R. Marchand, Woerly, S., Bertrand L. et al. Evaluation of two cross-linked collagen gels implanted in the transected spinal cord // Brain Res. Bull. - 1993. - V.30. - №3-4. - P.415-422; Marchant, R., A. Hiltner, C. Hamlin et al. In vivo biocompatibility studies. 1. The cage implant system and biodegradable hydrogel // J. Biomed. Mater. Res. - 1983. - V.17. - P.301-325; Motoyuki S., Kazuhiro I., S. Akiyoshi et al. Long-term culture of primary rat hepatocytes with high albumin secretion using membrane-supported collagen sandwich // Cytotechnology. - 1993. - V.11. No. 3. - P.213-218).

Gel Spherogel" has the following characteristics:

The pore size~100-400 µm
The amount of coupled water~32,8±0,5 wt.%
Swelling (in water)~86,6±3,0 wt.%
The time of complete resorption~ from 3-4 weeks to 6-9 months

Heterogeneous system hydrogel contains collagen in the form of microparticles (~35-300 µm) in the collagen solution.

Physico-chemical, mechanical and technological properties "Spherogel" make this biopolymer is very attractive to develop time frames for hybrid bioisosteric bodies, as have the following properties (Volova T.G., Sevastianov V.I., shyshatskyy H. Polyoxyalkylene - biorstwami polymers for medicine: Monograph. 2nd ed., more. and reprocessing. - Krasnoyarsk, Platina 2006. - 288 S.):

- versatility (simultaneously performs the functions of the frame, substrate and nutrient medium for cell cultures);

mechanical strength and elasticity sufficient to surgical manipulations;

- biocompatibility on the protein and cellular levels;

- the ability to stimulate the proliferation and differentiation of cells;

- porosity (size of micropores 100-400 μm), providing the processes novas is ularization;

- the possibility of sterilization standard ways without changing their medico-technical properties;

- ability to biodegradation (from 3-4 weeks to 6-9 months).

Used gels were disposable syringe volume: 2.0 ml, which were placed in a double sterile surgical package. Product "STERILE" (sterilized radiation method).

5). Transplantation of heterogeneous gel, with immobilizerturning by autologous liver cells and progenitor cells in the bone marrow in the body;

Transplantation of heterogeneous gels with immobilizerturning by autologous liver cells and autologous progenitor cells in the bone marrow provide patients with hepatic insufficiency by injection into the liver parenchyma and/or in the mesentery of the small intestine for 3-4 days after hepatectomy. For which we used the gel with immobilizerturning cells that were kept in the syringe. Moreover, when transplanted into the liver parenchyma carried out by performing at least one injection of the gel with immobilizerturning on the cells. When transplanted into the mesentery of the small intestine carry out at least one injection of the gel with immobilizerturning on the cells. When transplanted into the liver parenchyma and the mesentery of the small intestine carry out, respectively, at least one in which eccii gel with immobilizerturning on the cells in each of these areas.

6). The use of anticoagulants and antiplatelet agents;

After transplantation gel with immobilizerturning by autologous liver cells and autologous 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, for example, trental based 45 mg/m2body surface area every 12 hours for 30 to 90 days.

The proposed transplant for the treatment of liver failure consists of heterogeneous biocompatible biodegradable gel and planted him autologous progenitor cells from the bone marrow and autologous 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 pathologically and the modified liver, Byull. 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., Plekhanov A. N. 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.l04. - 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 autologous progenitor cells in 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 Gamboa. 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 lizirovania conducted twice 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). Carried out following the conventional method of culturing progenitor cells in the bone marrow.

Partially purified progenitor cells in the bone marrow, were sown for cultivation in the amount of 1.5-2.0 million cells/ml were Cultured at 37°C in CO2the incubator atmosphere of 5% CO2and 95% humidity for stock with a single change of environment on the third day.

3). The allocation of autologous liver cells was carried out according to traditional methods.

The allocation of autologous liver cells from resected area of the liver (4×4×2 cm in dogs and 1×1×0.5 cm in rats) produced by 3-fold the washing of the 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 from 3.0×108to 4.0×108hepatocytes on 15 g of liver tissue. The cell suspension contained: hepatocytes ~95÷98%, and ~5÷2% nepriskonowennij liver cells, which were identified by light microscopy. The separation of the pair is hemitonic and neprekonatelnych cells was not performed. A suspension of liver cells was concentrated to 1-2 ml of physiological solution

3). Landing (immobilization) autologous liver cells and autologous bone marrow cells in a heterogeneous gel.

Landing (immobilization) autologous liver cells and progenitor cells from the bone marrow to the gel also carried out according to traditional methods.

As a heterogeneous gel can be used, for example, a biomaterial, "Spherogel" made on the basis of collagen farm animals (70%) and collagen extract (30%)with the appearance of a thick jelly-like substance. With a pore size of ~ 100-400 μm, the time for complete resorption ~ from 3-4 weeks to 6-9 months.

Autologous liver cells and autologous 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 cells of bone marrow cells/ml Total cell suspension at a concentration of 2×106-15×106on 1 cm3gently mixed with a heterogeneous gel in a ratio of 1:1, until a uniform consistency was collected in a syringe and kept until the introduction in the liver and/or mesentery at 4°C for not more than 5 hours.

4). Transplantation gels with immobilizerturning by autologous liver cells and progenitor cells in the bone marrow in the body of animals:

A). Dogs exercised in the liver parenchyma for 3-4 days after modeling liver failure by resection of 50% of the liver tissue;

B). Dogs exercised in the mesentery of the small intestine on day 3 or 4 after modeling liver failure by resection of 50% of the liver tissue;

B). Dogs exercised in the liver parenchyma and in the mesentery of the small intestine on day 3 or 4 after modeling liver failure by resection of 50% of the liver tissue;

G). Rats in the liver parenchyma at 42-44 days after modeling chronic liver failure by the introduction of a 60% solution of CCl4(the first administration of 0.5 ml per 100 g mass, subsequent to 0.3 ml per 100 g body weight frequency - 2 injection per week).

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 of the Operation p is bodily under endotracheal anesthesia. Maintenance of anesthesia was performed with 1% Propofol 0.5 mg/kg/h and Kalipsola 2 mg/kg In rats was carried out by anesthesia allowance per 100 g body weight, were injected intramuscularly: Calypsol 10% - 2 ml, Xylazine 2% - 1 ml Atropine of 0.2 ml.

Total cellular material containing autologous liver cells and bone marrow at a concentration of 2×106-15×106on cm3and a ratio of 1:1, immobilized on a heterogeneous gel Spherogel, transplanted into the liver parenchyma and/or in the mesentery of the small intestine of animals.

The abdominal cavity is sutured in layers tightly. For the prevention of infectious complications intraoperatively animal was intravenously injected 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.

5). 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 gel 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, such as Trent is based 45 mg/m 2body surface area every 12 hours for 30 to 90 days.

Altogether, we conducted 18 experiments, with correction of acute liver failure under the proposed method was carried out 3 experiments and chronic liver failure - in 15 experiments.

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

Figure 1 presents the dynamics of the synthetic function of the liver (fibrinogen) 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 fibrinogen decreased 2.4 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 heterogeneous gel planted on him autologous progenitor cells in the bone marrow and autologous cells of the liver 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) is otologically liver cells and progenitor cells from the bone marrow, planted on heterogeneous gel, indicated more rapid recovery to baseline liver synthetic function, for example, to 5 days of transplantation, this figure was smaller than the original by 9.5%and in the control group 44.3%.

Figure 2 presents the dynamics of the synthetic function of the liver (APTT) in acute liver failure. Studies have shown that after resection of 50% of liver synthetic function of the liver 1 day after resection decreased, which resulted in an increase of APTT by 33.3%. Compared with the outcome on day 3 after resection indicators APTT increased 1.35 times, which indicates a significant functional impairment of liver cells. Then a gradual (and in the study group, almost to 5 days after transplantation), the return of these indicators to the original, and in the control group in the study period (18 days after the operation) this indicator (APTT) has not reached the source. This only happened to 28-32 days after the operation.

From the presented data it follows that until the transplantation of autologous liver cells and progenitor cells from the bone marrow, planted on heterogeneous gel, the dynamics of indicators characterizing the synthetic function of the liver was similar in both groups (control and experiment), however, research in the group has been created (experiment) recovery of liver synthetic function (fibrinogen and APTT) was faster and these figures were closer to the source.

Figure 3 90 days in the control group (without treatment by the proposed method) revealed large and small droplet fatty degeneration of hepatocytes; parenchymatous degeneration of hepatocytes; focal necrosis of hepatocytes (1). In the parenchyma rare lymphoid cell infiltration and proliferation of histoblasts and historical (2).

Coloring PAS, HC.×400.

Figure 4 90 days in the control group (without treatment by the proposed method) revealed hydropic degeneration of hepatocytes (3), focal necrosis (4).

Paint hematoxylin-eosin, HC.×400.

Figure 5 90 days in the study group (treatment according to the proposed method) revealed intact structure of the liver and its Molochnoe structure (5). The Central part of the slices with intact structures (5).

Paint hematoxylin-eosin, HC.×400.

Figure 6 90 days in the study group (treatment according to the proposed method) revealed the island implanted hepatocytes near the Central vein (6).

Paint hematoxylin-eosin, HC.×400.

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 autologous hepatocytes, detoxication is Noah and bioregulatory functions extension of the terms of their survival by activation of proliferation through joint intracorporeal injection of autologous 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 liver and/or 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 the device with cells. In addition, the use of autologous liver cells and progenitor cells from the bone marrow avoids the use of immunosuppressive therapy, and long-term viability allows you to maintain homeostasis.

Thus, the above data clearly indicate that correction of liver failure is much faster and more appropriately in the application of the proposed method of correction of liver failure. In addition, our proven long-term survival of liver cells and the appearance of newly formed vessels in the device after 90 days after transplantation animal gel with the autologous liver cells and autologous progenitor cells cost the th brain.

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, wherein the pre-exercise intake of autologous bone marrow cells and their cultivation in vitro, then taking autologous liver cells, perform landing freshly isolated liver cells and cultured bone marrow cells in heterogeneous biocompatible biodegradable gel with pore size of 30-500 μm and a total porosity of 50-98%, providing a total concentration of liver cells and progenitor cells from the bone marrow 2·106-15·106cells on 1 cm3heterogeneous gel and the ratio of progenitor cells from the bone marrow to the liver cells from 1:1 to 1:4, and the total amount of heterogeneous gel should be not less than 0.1 ml, and transplanted gel with liver cells and bone marrow in the liver parenchyma and/or the mesentery of the small intestine.

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

3. The method according to claim 1, characterized in that the culturing progenitor cells in the bone marrow is carried out for 7 days.

4. The transplant for the treatment of liver failure, including heterogeneous biocompatible biodegradable gel, with a total volume of 0.1 ml and the smallest linear dimension of at least 0.2 mm, a pore size of 30-500 μm, the total porosity 50-98% and planted on him autologous progenitor cells in the bone marrow after their cultivation in vitro and cultured autologous liver cells, and the concentration of liver cells and bone marrow 2·106-15·106cells on 1 cm3heterogeneous gel and the ratio of bone marrow cells to liver cells from 1:1 to 1:4.



 

Same patents:

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, pharmaceutics.

SUBSTANCE: invention relates to chemical-pharmaceutical industry, namely to creation of medication, which possesses hepatoprotective, detoxicating and regenerating action. Medication contains in its composition essential phospholipids, vitamin B1, vitamin B2, vitamin B6, vitamin E, extract of milk thistle, extract of artichoke and auxiliary substances. Claimed medication extends arsenal of hepatoprotective medications, which possess wide pharmacologic spectrum of action.

EFFECT: medication corresponds to polyvalency of disease pathogenesis, affects patient's organism as a whole as corrective system.

2 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: invention refers to veterinary science, namely to therapy of internal non-transmitting diseases, particularly to drugs for pig hepatosis prevention. The agent for pig hepatosis prevention contains the ingredients in the following relation: antihepatotoxic serum 0.9-1.15 titre units; antisplenotoxic serum 0.9-1.15 titre units; leukocyte plasma 90-110 embryo units; phenol 0.004-0.005 mg; physiologic saline up to 1 ml.

EFFECT: invention allows higher effectiveness of pig hepatosis prevention.

4 tbl

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: method involves per os administering Hepatosan at a dose of 0.8 g/day. The total treatment course is 2 months long.

EFFECT: high activity of 7-α hydroxylase; no adverse side effects observed.

3 tbl

FIELD: medicine, clinical pharmacology, restoring therapy.

SUBSTANCE: the suggested preparation is being a lipid fraction isolated out of a king crab's liver Paralithodes comtschatica that contains 10% polyunsaturated fatty acids, 10% alkyl-diacyl glycerides and the complex of natural bioantioxidants being of highly correcting, hypocoagulative and antioxidant properties. The preparation suggested enables to widen the quantity of food BAA of natural origin of lipid-correcting action at pronounced hypocoagulative and antioxidant properties based upon application of natural lipid complex. It could be applied as an efficient means of prophylaxis of cardio-vascular diseases and other pathologies, when lipid exchange is broken and which is accompanied with disorders of hemocoagulation and intensification of lipoperoxidation process.

EFFECT: higher efficiency.

1 ex, 4 tbl

FIELD: medicine.

SUBSTANCE: invention relates to biology and medicine, namely to experimental models of immunologic states, and can be used for estimation of anti-ergotypic response to vaccination with polyclonally activated cells. For this purpose vaccination with splenocytes, activated by means of anti-CD3 antibodies and interleukin-2, by subcutaneous injections 1 time per week during 4 weeks. After that response of delayed-type hypersensitivity (DTH) is induced by introduction of activated splenocytes under aponeurosis of animal paw pad. Anti-ergotypic response is estimated by degree of DTH development.

EFFECT: implementation of method provides possibility of experimental reliable estimation of anti-ergotypic response in vivo, induced by polyclonally activated cells, and estimate its connection with changes of autoimmune disease course, performing control over autoimmune responses.

1 dwg, 2 tbl, 3 ex

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