Method of isolating hepatocytes from birds of genus columba livia

FIELD: medicine.

SUBSTANCE: method includes opening of abdominal cavity, cannulating of v.portae, through which two-step perfusing is performed, mechanical processing of liver, purification of hepatocytes, filtration and differential centrifuging, preliminary incubation and inoculation of hepatocytes in Goryaev chamber, primary perfusing being performed with calcium-free Hanks solution, the secondary one being performed three-four times with Hanks solution, which contains calcium ions and 1 type collagenase in concentration 0.02-0.05% for 20 minutes at perfusate temperature 15-16°C; purification of hepatocytes in ficoll gradient is performed three-four times for 15 minutes on centrifuge with 1000 rev/min at temperature 20°C with addition of 0.5-1.15 M of saccharose, differential centrifuging is carried out with 700 rev/min with 2 minute exposition; preliminary incubation of hepatocytes is carried out at 37°C for 20 minutes.

EFFECT: invention makes it possible to increase percentage of output of viable hepatocytes of birds of genus Columba livia, and can be used for studying mechanisms of cell functioning, in diagnostics of viral infections, in production of vaccines, correction of functions of injured liver tissues.

3 tbl, 3 ex

 

The invention relates to biology and medicine and can be used for studying the functioning of cells, control of cellular processes such as proliferation and differentiation, to learn the basics of intercellular interactions and modification of metabolism in physiological norm and experiment in the diagnosis of viral infections to study the spectrum of sensitivity of hepatocytes of various animals to the virus, in biotechnological processes for the production of vaccines, replacement, repair, adjustment functions of the damaged liver tissue by implantation or transplantation grown in vitro hepatocytes.

To date we are not aware of how the selection of hepatocytes bird species Columba livia, which have characteristics in common with the claimed method.

The task of the invention is to develop a method that would ensure the allocation of hepatocytes bird species Columba livia with a high percentage yield of viable hepatocytes. It should be emphasized that a high percentage of viable hepatocytes is a necessary condition for successful biomedical research, and thus obtain significant (reliable) of the examined parameters.

The problem is solved due to the fact that the inventive method includes the t such signs, as: opening the abdomen, kanalirovanie v.portae through which implementing a two-stage perfezione, machining of the liver, treatment of hepatocytes, filtering and differential centrifugation, pre-incubation and seeding of hepatocytes in Petri dishes to determine the number of viable hepatocytes in the cell Goryaeva, with the primary perfezione liver spend Beccalli Hanks solution for cleaning the blood and removing calcium ions, and secondary perfezione liver spend three to four Hanks solution containing calcium ions and collagenase type I at a concentration of 0.02 to 0.05% in 20 min at the temperature of the perfusion solution 15-16°C, a three-to fourfold purification of hepatocytes in Pikalova gradient for 15 min in a centrifuge at 1000 rpm and 20°C with the addition of 0.5-1.15 M carbohydrates - sucrose, differential centrifugation is carried out at 700 rpm with an exposure of 2 min, and pre-incubation of hepatocytes was performed at 37°C for 20 minutes

The inventive method of allocation of hepatocytes bird species Columba livia is implemented as follows.

Under ether anesthesia the birds spend opening the abdomen, kanalirovanie v.portae, which is carried through the primary perfezione liver Beccalli Hanks solution for cleaning the blood and removing calcium ions, ZAT is a three-to fourfold conduct secondary perfezione liver solution Hanks, containing calcium ions and collagenase type I in ascending concentrations (from 0.02 to 0.05%) for 20 min at the temperature of the perfusion solution 15-16°C. Then, after removing Glassonby capsules and large blood vessels of the liver are crushed in penicillin vial with scissors into pieces weighing 1 to 2 g, which is transferred into the flask containing the medium Needle, which is then placed on the magnetic stirrer, for example type MS3000, Biosan, Latvia (Latvia), for 5 minutes. Followed by a three-to fourfold purification of the obtained tissue suspension in Pikalova gradient for 15 min in a centrifuge, for example, ARF-3, (Kyrgyzstan), at 1000 rpm and 20°C with the addition of 0.5-1.15 M of carbohydrates, such as sucrose, to remove cellular debris, blood cells, hematopoietic cells and large fragments. Then the precipitate resuspension in the incubation medium, and then the cell suspension was filtered through nylon filters into the flask and leave for pre-incubation for 20 min at 37°C on a shaker, for example of the type BS-010108-AK, UP to 12, Biosan, Latvia (Latvia), in order to remove debris of damaged cells.

To obtain population parenchymal cells are filtered through nylon filters and differential centrifugation, for example type VAC-601, for 2 min at 700 rpm and then the camera Goryaeva 1% solution Trypanosoma blue to determine icesto viable hepatocytes at a concentration of cells in the 3×10 6cells/ml Then the cell suspension is poured into Petri dishes, the bottom of which is placed a covering glass, pre-treated with 0.2% solution of gelatin (Sigma, USA). Incubated hepatocytes in F12 medium, which contained 0.2 mg/ml albumin (Sigma, USA) and 0.5 μg/ml insulin (Sigma, USA)and containing 40 μg/ml gentamicin, 10% fetal serum (PAA, Austria), 25 units/ml of factor LIF (Sigma, USA). Then the Petri dishes placed in a thermostat, for example of the type BS-010410-BAA, SP-100, Biosan, Latvia (Latvia), for culturing at 37°C.

Presented concrete examples of implementation of the proposed method.

Example 1. The influence of the fractional perfusion and temperature regimes on the percentage yield of viable hepatocytes birds Columba livia

The animal opened the abdominal cavity, then spent kanalirovanie v.portae, which is carried through the primary perfezione liver Beccalli Hanks solution for cleaning the blood and removing calcium ions.

Secondary perfezione liver is conducted according to established groups fractionally Hanks solution containing calcium ions and collagenase type I in ascending concentrations (from 0.02 to 0.05%). For experience has formed three groups of birds:

group 1 - perfusion was performed once with Hanks solution containing calcium ions and 0.05% collagenase type I, 5 min at room temperature;

group 2 - 10 min triple R is the target Hanks, containing calcium ions and collagenase type I in ascending concentration when the temperature of the perfusion solution 10-15°C;

3 - the group of 20 min three times with Hanks solution containing calcium ions and collagenase type I in ascending concentration when the temperature of the perfusion solution 15-16°C.

Table 1
The influence of the fractional perfusion and temperature regimes on the yield of hepatocytes (% viable cells)
no groupBirds of the species Columba livia
group 186
group 289
group 398

Table 1 confirms that for birds, the liver are characterized by a high density, fractional perfezione by collagenase three times for 20 min at the temperature of the perfusion solution 15-16°C (group 3) is optimal and provides the maximum percentage yield of viable hepatocytes in comparison with the first and second group.

Then the liver was crushed scissors, as a stage of mechanical treatment, the tissue suspension was placed on a magnetic stirrer, and then conducted a three-to fourfold purification of danilovici in Pikalova gradient 15 min in a centrifuge 1000 rpm at a temperature of 20°C with the addition of 0.5-1.15 M of carbohydrates, for example, sucrose, obtained cell suspension of hepatocytes through a combination of filtering and differential centrifugation for 2 min at 700 rpm, the subsequent pre-incubation of hepatocytes spend on a shaker in an incubator at 37°C for 20 minutes, the cell suspension of hepatocytes in the amount of 3 ml (3×106hepatocytes) are poured into Petri dishes, the bottom of which are cover glass coated with gelatin. Then the camera Goryaeva 1% solution Trypanosoma blue to determine the number of viable hepatocytes at a concentration of cells in the 3×106cells/ml Then the Petri dishes are placed in the incubator for culturing at 37°C.

Example 2. Obtaining a cell suspension and clearance in Pikalova gradient with addition of carbohydrates in birds Columba livia

The animal opened the abdominal cavity, then spent kanalirovanie v.portae, which is carried through the primary perfezione liver Beccalli Hanks solution for cleaning the blood and removing calcium ions. Secondary perfezione spend Hanks solution containing calcium ions and collagenase type I in ascending concentrations (from 0.02 to 0.05%) for 20 min at the temperature of the perfusion solution 15-16°C. Next, the liver was crushed scissors, as a stage of mechanical treatment, the obtained tissue suspension was placed on a magnetic stirrer.

group 1 - cleaning Pikalova the single gradient;

group 2 - cleaning Pikalova gradient three-to fourfold;

group 3 - cleaning Pikalova gradient three to four-fold with the addition of 0.5-1.15 M of carbohydrates.

Table 2 shows that the addition of carbohydrates and three-to fourfold purification in Pikalova the gradient increases the yield of viable hepatocytes in group 3 by 19% compared with the first group and 11% in comparison with the second. We offer carbohydrate metabolic correction contributes to the preservation and stabilization of membranes and energy metabolism of mitochondria, ensuring the preservation of viable hepatocytes.

Table 2
The effect of fractional purification in Pikalova gradient with addition of carbohydrates on the percentage yield of hepatocytes (% viable cells)
no groupBirds of the species Columba livia
group 178
group 286
group 397

Next received cell suspension hepatocytes PU is eating a combination of filtering and differential centrifugation for 2 min at 700 rpm, subsequent pre-incubation of hepatocytes on a shaker spend 20 minutes in a thermostat at a temperature of 37°C. the cell suspension of hepatocytes in the amount of 3 ml (3×106hepatocytes) are poured into Petri dishes, the bottom of which are cover glass coated with gelatin. Then the camera Goryaeva 1% solution Trypanosoma blue to determine the number of viable hepatocytes at a concentration of cells in the 3×106cells/ml Then the Petri dishes placed in a thermostat for culturing at 37°C.

Example 3. Getting parenchymal cell suspension birds Columba livia

The animal opened the abdominal cavity, then spent kanalirovanie v.portae, which is carried through the primary perfezione liver Beccalli Hanks solution for cleaning the blood and removing calcium ions. Secondary perfezione spend Hanks solution containing calcium ions and collagenase type I in ascending concentrations (from 0.02 to 0.05%) for 20 min at the temperature of the perfusion solution 15-16°C. Next, the liver was crushed scissors, as a stage of mechanical treatment, the obtained tissue suspension was placed on a magnetic stirrer. Conducted clearing of tissue suspended in Pikalova gradient three-four times for 15 min in a centrifuge 1000 rpm at a temperature of 20°C with the addition of 0.5-1.15 M of carbohydrates, such as sucrose.

For gaining the parenchymal cell suspension were experimentally selected modes differential centrifugation:

I - 5 min at 200 rpm;

II mode 5 min at 700 rpm;

III mode - 2 min at 700 rpm

From table 3 it can be seen that the highest yield of viable hepatocytes was observed at 3 mode (2 min at 700 rpm) - 97%, which is optimal, since this regime is achieved minimum mechanical effect on hepatocytes and differentiated parenchymal division and neverending liver cells.

Table 3
The effect of different regimes of differential centrifugation on the percentage yield of hepatocytes (% viable cells)
no groupBirds of the species Columba livia
group 174
group 283
group 397

Subsequent pre-incubation of hepatocytes on a shaker spend 20 minutes in a thermostat at a temperature of 37°C. the cell suspension of hepatocytes in the amount of 3 ml (3×106hepatocytes) are poured into Petri dishes, the bottom of which are cover glass coated with gelatin. Then the camera Goryaeva 1% solution of the trip is the new blue to determine the number of viable hepatocytes at a concentration of cells in the 3×10 6cells/ml Then the Petri dishes placed in a thermostat for culturing at 37°C.

The obtained culture is a morphologically homogeneous population of cells that retain a stable karyotype, free from extraneous agents. Monoclona culture has the appearance of a morphologically homogeneous layer of cells attached to the glass and covered with a transparent nutrient medium red-orange color. Work with the drug in sterile conditions. Before opening bottles with cells pay attention to the integrity and concentration of ions in the medium (pH) is not lower than 7.0.

Thus, the inventive method provides for the selection of hepatocytes bird species Columba livia with high (up to 97) the percentage yield of viable hepatocytes.

Isolation of hepatocytes bird species Columba livia, including opening the abdomen, kanalirovanie v.portae through which implementing a two-stage perfezione, machining of the liver, treatment of hepatocytes, filtering and differential centrifugation, pre-incubation and seeding of hepatocytes in Petri dishes to determine the number of viable hepatocytes in the cell Goryaeva, with the primary perfezione liver spend Beccalli Hanks solution for cleaning the blood and removing calcium ions, and secondary perfezione liver Prov is completed with a three-to fourfold solution Hanks, containing calcium ions and collagenase type 1 at a concentration of 0.02 to 0.05% in 20 min at the temperature of the perfusion solution 15-16°C, a three-to fourfold purification of hepatocytes in Pikalova gradient for 15 min in a centrifuge 1000 rpm at a temperature of 20°C with the addition of 0.5-1.15 M sucrose, differential centrifugation is carried out at 700 rpm with an exposure of 2 min, and pre-incubation of hepatocytes was performed at 37°C for 20 minutes



 

Same patents:

FIELD: medicine.

SUBSTANCE: described is method of cell analysis by means of biochip, containing immobilised molecules of substances, able of binding with molecules, which are on the surface of cells, includes incubation of biochip with suspension of cells. Then, washing of biochip from nonspecifically bound cells is carried out. After that, fixation and staining of cells, reading of results and estimation of quantity of cells, which bound in one or several parts of biochip of the given area, are performed. In conclusion, analysis of the image of bound cells is carried out. Before carrying out fixation from biochip surface excess of liquid is removed, without permitting it to dry.

EFFECT: improvement of technology.

4 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: method includes cultivation of pluripotent stem cells in culture medium, which does not contain substance, stimulating activation of canonical signal way Wnt, during period of time between beginning of differentiation induction and 24 hours before period of increased expression of genes of canonical Wnt. After that carried out is cultivation of pluripotent stem cells in culture medium, which contains substance, stimulating activation of canonical signal way Wnt, during period of time from 24 to 96 hours, starting from 24 to 0 hours before period of increased expression of genes of canonical Wnt.

EFFECT: method makes it possible to induce cardiomyocyte differentiation efficiently and selectively.

15 cl, 15 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: method includes cultivation of pluripotent stem cells in culture medium, which does not contain substance, stimulating activation of canonical signal way Wnt, during period of time between beginning of differentiation induction and 24 hours before period of increased expression of genes of canonical Wnt. After that carried out is cultivation of pluripotent stem cells in culture medium, which contains substance, stimulating activation of canonical signal way Wnt, during period of time from 24 to 96 hours, starting from 24 to 0 hours before period of increased expression of genes of canonical Wnt.

EFFECT: method makes it possible to induce cardiomyocyte differentiation efficiently and selectively.

15 cl, 15 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: method of cryoconservation of multipotent mesenchymal stromal cells (MMSC) includes preparation of culture mixture in culture medium, mixing cell culture in culture medium with cryoprotector, step-by-step controlled cooling and freezing mixture of culture medium with cryoprotector to storage temperature and further storage of frozen mixture at low temperatures. As cryoprotector for mixture saturation used is xenon gas, introduced till saturation is achieved into prepared mixtute of cell culture MMSC in DMEM medium, which is in open cryo test tubes by blowing culture medium with xenon at 0°C with further cooling and formation of primary monolith of culture medium with cryoprotector, directed later at cooling to storage temperature.

EFFECT: invention ensures preservation of cells in cryopreservation.

4 cl, 2 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: described is method of obtaining population of stem cells, which come from dental follicle of human, named FENC (coming from follicles stem cells of embryonic nerve crest), which include: a) collection of follicle sac in sterile conditions, splitting, growing and grafting of initial culture; b) optional amplification; c) sorting out on FAC - sorter by the following stem characteristics: SSEA-4, TRA 1-60, TRA 1-81, OCT-4+; d) analysis of RNA with respect to positivity by transcriptional factors Nanog and Rex-1.

EFFECT: invention makes it possible to obtain homogenous population of stem cells.

10 cl, 6 ex

FIELD: medicine.

SUBSTANCE: method provides the following stages: (a) preparing a matrix, (b) preparing isolated hepatocytes, (c) inoculating a matrix of density 2 to 4x103 mm-2 with hepatocytes, (d) leaving the cell-coated matrix for 10-180 minutes so that to enable the cells to adhere to the matrix, (e) washing the nonadherent cells from the cell-coated matrix, (f) leaving the cell-coated matrix for max. 180 minutes, and (g) freezing the cell-coated matrix in a freezing medium, (h) storing the frozen cell-coated matrix, (i) defrosting the frozen cell-coated matrix, (j) washing the nonadherent cells from the cell-coated matrix, (k) coating the defrozen cell-coated matrix with a layer of a second matrix, and (1) reculturing the cells built-in between the matrixes.

EFFECT: invention allows providing an increased percentage of viable hepatocytes in the sandwich-type cultures.

12 cl, 4 dwg, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method involves increasing biomass of human embryo stem cells of the feeder-less hESKM-05 with the use of a base mTeSR medium in the bottles coated with 0.1 % gelatin. An increasing procedure is enabled with the use and daily change of a base coDMEM medium containing 10% serum substitute SR, 100 mcg/ml of kanamycin sulphate, 1 mM L-glutamine, 4 ng/ml of a base fibroblast growth factor (bFGF), 1 mM essential amino acids. A conditioned medium is produced from a culture of mice embryo neuronal cells. The increased biomass is transferred by means of 0.5 % collagenase in bottles containing a prepared collagen-chitosan matrix in the conditioned medium or in a complete nutrient medium with neuronal factor N2 added. The medium is changed every three days.

EFFECT: invention allows producing the neuronal matrix suitable for direct transplantation.

16 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method involves preparing a biological referent material on the basis of a biological material of animal origin, after alimentary introduction to an animal of water-soluble lead or mercury or cadmium salts before ensuring in the biological materials of the certain lead or mercury or cadmium concentrations. Thereafter, the animals are killed by decapitation. Further, the biological material is sampled with an aliquote being analysed for the preset concentrations of toxic metals, then the biological material is packed and lyophilised.

EFFECT: invention allows improving diagnosing toxic metal poisonings and providing higher accuracy of determination of the toxic metal concentrations.

11 cl, 3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method includes increase of biomass of human germ stem cells of non-feeder line hESKM-05 with application of main medium mTeSR in flasks covered with matrigel. After that, performed is subcultivation by means of 0.05% dispase solution. Then, in order to form germ stem cells of embrioid bodies, by means of dispase cells are transferred into "коДМЕМ" medium with addition of 10% substituent of SR serum, 100 mcg/ml of sulfate canamicine, 1mM of L-glutamine solution and 1 mM solution of essential amino acids, as well as 2 mcg/ml solution of 5-asa-2-seoxycitidine or 2 mcM of sodium butirate solution and stimulated for 3 days. After that retransmission onto prepared collagen-chitosane matrix and cultivation in nutritional medium "коДМЕМ" with addition of 1 mM solution of essential amino acids, 1 mM of L-glutamine, 10% substituent of SR serum, 10-7 M of retinoic acid and 10 ng/ml of ascorbic acid are performed with replacement of solution every three days.

EFFECT: invention makes it possible to obtain cardiomyicytal matrix suitable for direct transplantation.

7 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: stem cells of an adult human recovered from subcutaneous fat eye tissues are induced to differentiate in insulin-secreting cells in a medium containing cytokines and growth factors, including the B27 additive, fibroblast-2 growth factor, epidermal growth factor, nicotinamide, glucagon-like peptide-1, active A, insulin-like growth factor and betacelluline. For a cultivation process, high-concentrated glucose is replaced for low-concentrated glucose.

EFFECT: invention enables producing cells synthesising considerable amounts of insulin and C-peptide.

3 cl, 8 dwg, 2 tbl, 9 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to a composition including alginate containing high concentration of mannuronic acid and a polycation of a polydispersity index less than 1.5. The composition is especially suitable for manufacturing of biologically compatible microcapsules containing live cells for allo- or xenografting.

EFFECT: microcapsules are more durable and can keep their structural and functional integrity for a long period of time as compared with common alginate microcapsules.

57 cl, 2 ex, 6 tbl

FIELD: medicine.

SUBSTANCE: inventions relate to medicine, namely to cell transplantology, and deal with method and transplant for treatment of liver failure. For this purpose autologous progenitor cells of bone marrow are isolated and cultivated in vitro. Also realised is sampling of autologous liver cells. After that, immobilisation of autologous liver cells and progenitor cells of bone marrow on carrier - biodegradable biocompatible three-dimensional matrix is performed. After that, transplantation of carrier with cells is performed by its introduction into mesentery of small intestine. Transplant includes biodegradable biocompatible three-dimensional porous matrix with pore size 2-500 mcm and total porosity 50-97%, ensuring total concentration of liver cells and progenitor bone marrow cells 2×106-15×106 cells per 1 cm3 of matrix and ratio of progenitor bone marrow cells to liver cells from 1:1 to 1:4. Total volume of matrix constitutes not less than 0.05 cm3, its smallest linear size being not less than 0.2 mm.

EFFECT: inventions make it possible to improve results of liver failure treatment due to prolongation of terms of hepatocyte survival and activisation of their proliferation, creation of conditions for growing of vessels into creates carcass, diffusion of nutrients, oxygen and factors of tissue differentiation, make it possible to avoid application of immunosuppressive therapy.

6 cl, 6 dwg

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

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: method involves introducing bio-organic preparation into patient organism. The preparation is introduced at a daily dose of 2 ml intramuscularly during 3-4 weeks.

EFFECT: enhanced effectiveness of treatment.

2 dwg

Up!