RU2325921C2 - Method of transplant production for medical treatment of dog's and cat's insular diabetes and method of medical treatment of dog's and cat's insular diabetes - Google Patents

Abstract

FIELD: medicine; veterinary science.

SUBSTANCE: method of transplant production includes pancreas extraction, mechanical milling, treatment with dispersion solutions, repeated milling, rinsing with Hanks solution, placement to culture medium and release of β-cells. 26±2-days rabbit's foetus or 56±3-days cat's or dog's foetus is used to perform pancreas extraction. Repeated milling of pancreas is carried out to 0.1-1.0 mm, culture nutrient medium is added with 5-10% fetal calf serum, and release of β-cells is executed by culture medium change every 3-4 days during 10-14 days of cultivation. After that produced β-cells are washed of nutrient medium and serum with Hanks solution and placed into sterile physiologic saline. Method of insular diabetes treatment includes pancreas β-cells transplantation. Transplant is produced as described above. Dogs and cats take 5-7 injections of transplant dosed 0.2 ml directly through abdominal wall to parenchyma of left lobe of liver accompanied by ultrasonography. Invention enables to transplant β-cells without immunosuppression therapy of recipient, perform xenogenic (interspecific) transplantation (rabbit - dog, rabbit - rat) without immunosuppressant and transplant rejection.

EFFECT: interspecific transplantation without immunosuppressant and transplant rejection.

3 cl, 2 tbl

Description

The present invention relates to the field of veterinary medicine and cell biotechnology, in particular to the production of pancreatic material for transplantation to animals suffering from diabetes mellitus, and a method for the treatment of diabetes mellitus by transplantation.

Currently, the problem of treating diabetes in humans is relevant, however, this problem is also acute for animals of pedigree and productive value, therefore, the development of effective methods for the treatment of diabetes of animals is relevant.

A known method of obtaining cellular material containing β cells from mammals for transplantation. To do this, the resulting pancreas is crushed, treated with dispersing solutions of proteolytic enzymes (collagenase, trypsin) and, after resuspension, islets of β-cells are placed in a nutrient medium with the addition of 5-10% fetal bovine serum [1].

There is also known a method for producing a transplant containing β-cells of the pancreas for transplantation to people with diabetes mellitus and a method of treating people with diabetes by transplantation. At the same time, a pancreas of a newborn rabbit, human fetus, or other mammal is used for transplantation in patients with diabetes mellitus. The gland is washed in the Hanks solution with the addition of antibiotics, cut into fragments and the culture mattresses are inoculated with the obtained material, adding culture (growth) medium and mammalian serum, and they provide a mode in which β-cells migrate freely from cultured pancreatic microfragments to the bottom of the culture mattress why incubation is carried out at a temperature of 35-40 ° C for a period of two days to six weeks with a single or multiple decrease in temperature to 4-25 ° C and exposure at zhennoy temperature as well as the temporary one-time or periodic decrease in serum concentration of β- cells are then collected from the bottom of the mattress and the culture material is prepared for transplantation.

For the treatment of diabetes mellitus, people are transplanted with material containing pancreatic β-cells obtained using the phenomenon of β-cell migration, while the material can be transplanted into various organs and tissues: intramuscularly, in particular, into the rectus abdominis muscle, into the liver ( directly into the parenchyma or through the portal vein or its branches), into the pulp of the spleen, into the splenic vein, into the cavity of the peritoneum, into the specially created muscle pocket, into the large omentum [2].

The objective of our research was to obtain a transplant from animals containing pancreatic β-cells with subsequent transplantation to animals with diabetes mellitus, aimed at normalizing blood glucose levels, reducing the dose of exogenous insulin administered and preventing the accompanying course of diabetes mellitus vascular complications.

This was achieved due to the fact that the pancreas of the fetuses of animals were used as material at the gestational dates - 26 ± 2 days for rabbits, 56 ± 3 days for dogs, 56 ± 3 days for cats. The extracted pancreas is washed with Hanks solution, mechanically crushed, treated with dispersing solutions, crushed to a size of 0.1-1 mm, washed with Hanks solution and placed in a culture medium supplemented with 5-10% fetal bovine serum; then every 3-4 days, the medium is changed until a pure culture is obtained - 10-14 days, after which the obtained β-cells are washed from the nutrient medium and serum with Hanks solution and placed in sterile physiological solution; the quality of the graft is judged by lowering the level of glucose in the blood of a sick animal.

The resulting transplant is administered to animals with diabetes mellitus, mainly dogs and cats, directly through the abdominal wall into the parenchyma of the left lobe of the liver of 5-7 injections of 0.2 ml of suspension, under the supervision of an ultrasound scan; the results of the recovery of a sick animal are judged by lowering the level of glucose in the blood to a physiological norm.

The essence of the proposed method for obtaining a transplant is as follows: the extracted pancreas is placed in a Hanks solution with antibiotics. Remove the capsule, interlayers of connective tissue with blood vessels and the main branches of the excretory ducts. The decapsulated pancreas is placed in a deep watch glass and cut into fragments 2-3 mm in size. The ground material is washed with a Hanks antibiotic solution. After that, the fragments are poured with a 0.1-0.3% collagenase solution in Hanks 'solution at room temperature for 10-15 minutes, washed with Hanks' solution and thoroughly ground with eye scissors into microfragments 0.1-1 mm in size. The material is washed with 199 medium with 5-10% fetal serum and placed in a culture mattress. Change of environment 1 time in 3-4 days for 10-14 days.

A method of treating animal diabetes mellitus is as follows - before transplantation, the patient animal is given insulin therapy aimed at normalizing the level of glucose in the blood, 3 days before the transplantation of β-cells, the patient is prescribed therapy with anticoagulant agents. Before transplantation, the skin of the animal is treated standard for ultrasound, shaved, treated with an antiseptic, moisturized. General anesthesia and immobilization of the animal is not carried out. The graft, which is a suspension of β-cells in physiological saline, is collected in a syringe with a long needle with a mandrel for spinal puncture. A puncture of the abdominal wall is carried out to the left of the white line at the level of the end of the xiphoid process of the sternum under ultrasound control. The needle transdermally and through the muscle abdominal wall enter the parenchyma of the left lobe of the liver and after removal of the mandrel, the transplant is injected in 0.2 ml portions at 5-7 points. Immunosuppressive therapy after transplantation is not carried out. The control of the results of transplantation is evaluated by lowering the level of glucose in the blood to the physiological norm and, accordingly, by reducing the dose of insulin administered.

Specific examples are shown in the tables.

Table 1.
Changes in glucose levels in clinical patients, expressed in mmol / l Before transplantation Day after transplantation -one one 7 fourteen thirty 60 90 120 150 180 210 240 270 300 330 360 Stuff. 19.3 19 5.2 4.4 4.2 - 3.9 - - - - - - - - - - Cat "M" 44.3 7.8 6.4 4.2 3,7 6.6 4.4 4.1 3.8 3,7 4.7 4.6 3.9 4.6 4,5 5.8 4.1 English setter 32.6 33 twenty 7.2 - 8 22 eleven 5.8 31 eleven 3.3 2.7 - - - - Earl.Setter 26.7 9.7 eleven 4,5 4.7 4.8 5.3 4.8 4.8 4.8 4.3 4.3 4.3 - - - - Cat "K" 20.9 5.1 4.1 5.7 4.1 5.2 4,5 - - - - - - - - - - Risen. 21.3 12 fifteen twenty eleven 16 8.3 13 - - - - - - - - - Persian 18.8 9.7 5 4.7 10 7.7 - - - - - - - - - - -

Table 2.
Changes in the doses of exogenous insulin administered in clinical patients are expressed in μIed Day after transplantation -one one 7 fourteen thirty 60 90 120 150 180 210 240 270 300 330 360 Stuff. twenty 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Cat "M" 10 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 English setter 44 44 twenty eighteen 16 12 eighteen fourteen twenty fourteen twenty fourteen Earl.Setter thirty thirty thirty 16 0 0 0 0 0 0 0 0 Cat "K" fifteen fifteen 3 9 12 7 6 5 Risen. 40 40 fifteen 40 28 thirty 36 Persian twenty eighteen eleven eighteen 23

The technical significance of the proposed method for obtaining a transplant is as follows:

- the resulting material containing β-cells of the pancreas of animals does not have “mature” species-specific antigens, which allows transplantation of these cells without immunosuppressive therapy of the recipient;

- due to the lack of “recognition”, it allows xenogenic (interspecific) transplantation of material (rabbit - dog, rabbit - cat) without the use of immunosuppressants and without transplant rejection.

The proposed method for the treatment of diabetes of animals allows:

- non-invasively transplant the β-cells of the pancreas of animals transdermally, under ultrasound control into the liver parenchyma, without general anesthesia, without immobilizing the animal, without laparotomy;

- due to transplantation of β-cells of the pancreas of animals, normalize the level of glucose in the blood of sick animals and, accordingly, significantly reduce the dose or completely refuse to administer exogenous insulin;

- to prevent the development of vascular complications associated with diabetes mellitus (nephropathy, angiopathy, retinopathy), leading to irreversible changes in the kidneys, blindness, and vascular disorders.

The proposed method for producing a transplant and a method for the treatment of diabetes mellitus in animals have been tested by us in 2005-2006. with positive results in the Department of Cellular Biotechnology, All-Russian Research Institute of Experimental Veterinary Medicine named after Ya.R. Kovalenko RAAS, on the basis of the veterinary clinic "Biocontrol" Russian Cancer Research Center. N.N. Blokhin RAMS and on the basis of the Moscow ambulance veterinary care "Avers-Vet."

The proposed method of treatment will find application in the field of veterinary medicine for the treatment of diabetes and will save high-value animals.

Information sources.

1. Petropavlovskaia M., Rosenberg L. Identification and characterization of small cells in the adult pancreas: potential progenitor cells? Cell Tissue Res. 2002 Oct; 310 (1): 51-8.

2. Patent RU 2135193 C1, A61K 35/39, 1999.

Claims (2)

1. A method of obtaining a transplant for the treatment of diabetes in dogs and cats, including the extraction of the pancreas, its mechanical grinding, treatment with dispersing solutions, re-grinding, washing with Hanks solution, placement in a culture medium and isolation of β-cells, characterized in that for extraction pancreas use 26 ± 2-day-old fruits of rabbits or 56 ± 3-day-old fruits of cats or dogs, repeated grinding of the pancreas is carried out to a size of 0.1-1.0 mm, in a culture medium 5-10% fetal bovine serum is obtained, and β-cells are isolated by changing the culture medium every 3-4 days for 10-14 days of cultivation, after which the obtained β-cells are washed from the nutrient medium and serum with Hanks solution and placed in sterile saline. 2. A method of treating diabetes mellitus in dogs and cats, including transplantation of pancreatic β-cells, characterized in that the transplant is used according to claim 1, which is administered to dogs by 5-7 injections in a dose of 0.2 ml and cats directly through the abdominal wall into the parenchyma of the left lobe of the liver under ultrasound guidance.