RU2204399C1 - Method for pathogenetic treatment of chronic hepatic diseases in experiment - Google Patents

Abstract

FIELD: medicine, experimental gastroenterology. SUBSTANCE: one should use suspension of fetal cells of liver and/or spleen and/or thymus at the quantity of 1 ml at cellular dilution of 3-5•10⁵. Before introduction, suspension should be twice irradiated with He-Ne laser per 2 min at 5- min-long interval. Power density of radiation is 75 mW/sq. cm. Donor of fetal organs should be subcutaneously injected with 50%-oily solution of carbon tetrachloride 16 h before fetal organs' removal at the quantity of 0.4 ml/100 g body weight. The present method increases functional activity of fetal cells. EFFECT: improved hepatic regeneration processes. 2 ex

Description

 The invention relates to medicine, in particular to experimental gastroenterology, and can be used to treat chronic liver disease in an experiment.

 A known method for studying the enzymatic activity of cytochrome P450 inside transplanted small pieces of the liver of the embryo. Hepatocytes were collected on the 20th day of pregnancy of rats and transplanted into the spleen of an adult recipient (see Kato K. et al. Enzymatic activity and expression of cytochrome P450 inside embryonic hepatocytes transplanted into rat spleen. "/ Internet / / Yahoo. Department of Medicine and Gastrointestinal Intestinal Surgery, Central Library of New York College of Medicine, Valhalla, USA, September 1999).

 Microscopic examination of recipient spleens at 4-10 weeks after transplantation revealed many hepatocytes with cord-like structures in the red pulp. These results demonstrated that hepatocytes can be transplanted into the spleen of the donor and subsequently grow in the spleen.

 However, the disadvantage of this method is the risk of necrosis of transplanted tissues due to the low probability of vascular invasion, impaired hemocirculation in the spleen, which can impair the function of both the liver and the spleen itself. This method is not physiological, as the transplant is performed in the spleen, and not in the liver.

 In addition, it is possible at a later date to absorb the basis of a portion of the transplanted material.

 Transplanted tissue pieces also have low functional and regenerative activity.

 A known method of transplantation of embryonic liver cells in an experiment, selected as the closest analogue, involving the injection of a suspension of fetal cells (see patent RU 2153344, Mcl A 61 K 33/407, A 61 P 43/00, publ. 27.07. 2000).

 According to the authors, the method allows to expand the functionality of the embryonic tissue, increase the responsiveness of the liver tissue and reduce the rejection reaction.

 The method, according to the authors, can be used for degenerative-dystrophic lesions of the liver.

 However, as the authors themselves note, positive results when using this method can be observed only 10-20 days after the experiment.

 Thus, the problem to which this invention is directed is to increase the functional activity of the transplanted remaining cells, achieve a positive result in a shorter time and, as a result, reduce the time of the experiment.

This task is achieved by the fact that in the known method for the pathogenetic treatment of chronic liver diseases in an experiment involving the injection of a suspension of fetal cells according to the invention, a suspension of fetal liver cells and / or spleen and / or thymus isolated from donor fetal organs is used for administration, received 16 hours before their extraction, a subcutaneous injection of a 50% oil solution of carbon tetrachloride in an amount of 0.4 ml per 100 g of weight, additionally before administration under irradiated twice in two minutes with a break of 5 minutes by irradiation with helium-neon lasers at a radiation power density of 75 mW / cm ² , while the amount of suspension introduced is 1 ml with cell dilution (3-5) • 10 ⁵ .

The applicant experimentally established that it was precisely the introduction into the recipient's organism of a suspension of fetal liver cells, and / or spleen, and / or thymus, moreover, isolated from the fetal organs of a donor who received subcutaneous injection of a 50% oil solution of carbon tetrachloride 16 hours before their extraction the amount of 0.4 ml per 100 g of weight and the additional before administration two times two minutes with a break of 5 minutes irradiation of this suspension with a helium-neon laser at a radiation power density of 75 mW / cm ² , with the suspension of 1 ml with a cell dilution (3-5) • 10 ⁵ , which contributes to a significant increase in comparison with all known methods, the functional activity of transplanted fetal cells, regenerative ability and, as a result, a significant acceleration of the process of regeneration of the damaged liver.

 This is because it is precisely due to laser irradiation (helium-neon) that the immunostimulatory properties of fetal cells are enhanced, which allows accelerating the regeneration of the damaged liver, which in turn accelerates the achievement of a positive result and reduces the time of the experiment.

 And the additional introduction into the donor organism of a 50% oil solution of carbon tetrachloride in the declared amounts and within the declared time period sharply activates the production of biologically active substances by the fetal tissues of the donor, which pass into the fatal cells implanted into the recipient and thereby further increase the regeneration process of the damaged liver.

 The set of essential features of the claimed object "method" has differences from the closest analogue and does not follow explicitly from the studied prior art, which indicates its compliance with the criteria of "novelty" and "inventive step".

 The claimed invention can find wide application in experimental medicine, which indicates its compliance with the criterion of "industrial applicability".

 The method in the experiment is as follows.

 Fetal liver tissue is taken from fetuses of female Wistar rats with a 20-day gestation period. 16 hours before the fetal tissue is removed from the body of the donor rat, it is injected with a 50% oil solution of carbon tetrachloride in an amount of 0.4 ml per 100 g of weight of the donor rat. This amount of hepatotropic poison does not have a toxic effect on the animal.

 The extracted fetal tissues are placed in saline.

 Then, using a homogenizer with a backlash that preserves the integrity of the cells, a mixture (suspension) of fetal cells is prepared.

On the basis of the obtained organ homogenates, a suspension of the remaining cells is prepared with a dilution of (3-5) • 10 ⁵ . The cellularity of the suspension is calculated using a Goryaev’s camera. Cell viability is assessed by staining their mixture with trypan blue.

 Then the suspension is placed in sterile tubes. A fiber is inserted into the tube, the diameter of which is chosen so that it fits tightly into the tube.

The fiber is installed at a certain distance from the surface of the suspension, without touching it, and is irradiated with a helium-neon laser twice in two minutes with a break of 5 minutes. Density laser power 75 mW / cm ² .

 The irradiated suspension is injected intraperitoneally to a biological object in an amount of 1 ml.

 Positive results (clinical, biochemical and histological) are observed on the 5-7th day.

 A suspension of fetal spleen and thymus cells and a mixture thereof, as well as a mixture of the remaining liver, spleen and thymus cells, is prepared in a similar manner.

 Animal experiments using this technique showed that the speed and quality of regenerative processes in the liver tissues significantly increase due to intracellular and cellular regeneration of hepatocytes, an increase in the number of multinuclear and coarse hepatocytes containing coarse chromatin, 1-3 nucleoli, as well as due to an increase in the proliferation of stellate reticulocytes.

 This is confirmed histologically and histochemically.

 Example 1. A biological object. Weight 200 g. Diagnosis: experimental cirrhosis of the liver. Stage of remission.

A suspension of fetal liver cells by dilution of 3 • 10 ^{5 was} prepared from fetal tissues of an animal donor, who received 0.8 ml of a 50% oil solution of carbon tetrachloride 16 hours before extraction of fetal organs.

After preparation, it was subjected to two-minute 2-minute intervals with a break of 5 minutes, irradiation with a helium-neon laser with a radiation power density of 75 mW / cm ² .

 The prepared and irradiated suspension was administered to a biological object in an amount of 1 ml intraperitoneally.

 Clinically positive results were observed 6 days after administration of the suspension.

 The animal is slaughtered after 3 weeks. The high regenerative ability of transplanted fetal cells was histologically and histochemically confirmed.

 Example 2. A biological object weighing 200 g. The diagnosis of chronic hepatitis. Stage of remission.

 The suspension preparation technology of Example 1. Differences: a suspension was prepared from fetal cells of the liver, and spleen, and thymus.

Dilution: 5 • 10 ⁵ . The amount of suspension introduced is 1 ml. The laser power density of 75 mW / cm ² .

 Positive results were observed 5 days after administration of a suspension of fetal cells.

 Results confirmed clinically, histologically and histochemically.

Claims (1)

A method for the pathogenetic treatment of chronic liver diseases in an experiment, which involves the injection of a suspension of fetal cells, characterized in that a suspension of fetal cells of the liver and / or spleen and / or thymus isolated from the fetal organs of the donor received 16 hours before extract subcutaneous injection of a 50% oil solution of carbon tetrachloride in an amount of 0.4 ml per 100 g of weight, additionally before administration, subjected to a double of two minutes with a break of 5 min reg cheniyu helium-neon laser radiation at a power density of 75 mW / cm ^2, wherein the amount of the slurry was 1 ml cell dilution 3-5 • ¹⁰ May.