RU2611205C1 - Method for obtaining of preparation based on stem cells selected from pigs spleen tissue, for prevention and treatment of infectious and non-infectious diseases of domestic and farm animals - Google Patents

Abstract

FIELD: medicine, pharmacy.

SUBSTANCE: invention refers to cell biotechnology and biopharmacology namely to preparation of the drug based on stem cells isolated from the spleen tissue of pigs, for the prevention and treatment of infectious and non-infectious diseases of domestic and farm animals. The invention may find application in veterinary medicine for treatment and prevention of a wide range of domestic and farm animals diseases. The method comprises administering of medium obtained from minced pig spleen tissue treated with 0.5% solution of metronidazole and 0.05% chlorhexine solution to the cell-culture medium, followed by disaggregation by 0.25% solution of trypsin and cell material culturing with cell concentrations of 1.5106-2.0106 per ¹ ml of growth medium Igla-MEM, supplemented with 10-15% fetal bovine serum in the presence of the Pinexide cell cultures growth stimulant, "Povin VM" preparation, which is composed of aqueous solution of polyethylene glycol, aqueous solution of PVP, commercial solution of Versen, solution tetracycline and commercial trypsin solution.

EFFECT: invention allows to extend the group of drugs based on stem cells for prevention and treatment of infectious and non-infectious diseases of domestic and farm animals with an expanded spectrum of application for treatment and structure recovery of lost animal organs.

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Description

The invention relates to the field of cell biotechnology and pharmacology, and relates to a method for producing a preparation based on stem cells for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals, as well as a method for isolating a stem cell culture from pig spleens. The invention may find application in veterinary medicine for the treatment and prevention of a wide range of diseases of domestic and farm animals.

Currently, pulmonary and gastrointestinal diseases of farm animals, which are ubiquitous and cause significant damage resulting from large waste and weight loss of young farm animals, have been supplemented by a number of other serious animal diseases: joint dysplasia in dogs and cats, microsporia, trichophytosis and others fungal diseases, mastitis, endometritis of farm and domestic animals, ophthalmic diseases, pathology of the cardiovascular system kyanite systems and others.

Among the drugs known in veterinary medicine, it is difficult to find a remedy that would simultaneously be therapeutic and stimulate the productivity of animals.

Known drugs for the treatment of respiratory and gastrointestinal infections of farm animals of bacterial etiology (patent No. 2053772, RU; patent No. 2214821, RU), as well as viral etiology (RF patent No. 2286171, CL. A61K 38/43, A61K 31/65, A61K 31/198, A61K 47/34, A61P 31/12, A61P 31/04, A61P 11/00, A61P 1/00, 2005, prototype), including an aqueous solution of polyethylene glycol, an aqueous solution of polyvinylpyrrolidone, a commercial solution of Versen, a solution of tetracycline and a commercial trypsin solution in the following ratio of components, vol.%:

15-20% aqueous solution of polyethylene glycol (MM 2000-6000). (PEG) 10-15 15-20% aqueous solution of polyvinylpyrrolidone (MM 280000-360000), (PVP) 10-20 0.01-0.02% Versen commercial solution 10-15 8-10% tetracycline aqueous solution 10-15 0.20-0.25% commercial trypsin solution (100-128 PIECES) Rest

turned out to be ineffective for the treatment of animal diseases such as joint dysplasia in dogs and cats, microsporia, trichophytosis and other fungal diseases, mastitis, endometritis of farm and domestic animals, ophthalmic diseases, pathology of the cardiovascular, respiratory, digestive systems and other diseases.

Productivity stimulants for farm animals are not widespread for a number of reasons, including the impossibility of using these pharmacological agents in bulk and with repeated use.

Currently, a new section is being formed in modern veterinary biomedicine - “cell therapy”, which allows using cell transplantation to make up for the insufficient functional activity of tissues and regenerate damaged organs. The function of updating and restoration of tissues in vivo is performed by stem cells, which are a pool of spare undifferentiated cell precursors of various types. In this regard, the use of stem cells is the most promising area of cell therapy, and work on the isolation of stem cells from human and animal tissues is of greatest relevance.

Different types of adult stem cells were distinguished - hematopoietic (blood cell precursors), neuronal (precursors of nerve tissue cells), mesenchymal (cells that can differentiate into tissue cells of mesenchymal origin, as well as other germ layers), etc.

Mesenchymal stem cells (MSCs) are distinguished by the relative simplicity of isolation and cultivation, the ability to proliferate over time in vitro, and a wide range of differentiation. In this regard, special attention is paid to methods of isolating MSCs from adult tissues, however, there is still no universal method for their preparation.

A known method of obtaining a biotransplant for the treatment of osteoporosis (RF patent No. 2265442, class A61K 35/28, 2004), consisting of MSC cells. In the process of obtaining MSCs, enzymatic disaggregation, filtration through a fine mesh sieve, centrifugation in Hanks saline solution, resuspension in DMEM / F12 growth medium containing 15% fetal calf serum and glutamine are carried out. Sowing is carried out on plastic Petri dishes when non-adherent cells are removed after 1 day. Incubation is carried out at 37 ° C in an atmosphere of 5% CO ₂ followed by trypsinization and reseeding culture. As a result of several passages, a cell culture of MSCs with the primary ability to osteogenic differentiation is obtained, but this culture is a heterogeneous cell population that is not characterized by phenotyping.

A known method for the isolation of mesenchymal stem cells (RF patent No. 2252252, class C12N 5/08, 2004) from human tissue, and can be used to treat a wide range of diseases. Human mesenchymal stem cells are isolated from adipose tissue, the decidual or amniotic membrane of the placenta, or the chorionic stroma of the placenta. Human tissues are crushed and treated with a collagenase solution in the Eagle medium in the Dulbecco modification, while the adipose tissue, decidual or amniotic membrane of the placenta is treated with collagenase type I, and the chorionic stroma of the placenta is treated with collagenase type IV. Then, the resulting suspension is purified from red blood cells using a lysis solution, followed by sequential filtration through filters with pore sizes of 100 and 10 μm.

A known method for the isolation of mesenchymal stem cells from human tissues (RF patent No. 2280462, class A61K 35/55, 2004), including crushing and enzymatic treatment of tissues with a collagenase solution in the environment of the Needle in the modification of Dyulbekko, purification from red blood cells using a lysis solution, followed by filtration the resulting suspension. Thymus is used as human tissue, type II collagenase is used for enzymatic treatment, and filtration is carried out sequentially through filters with pore sizes of 100 and 10 μm.

A known method of producing MSCs for implantation for the purpose of directed osteoregeneration (RF patent No. 2210352, class A61K 6/00, C12N 5/08, 2001), comprising isolating MSCs by disaggregating the source tissue of MSCs with subsequent resuspension of the suspension and culturing on growth medium.

Stem cells and their use in the treatment of many diseases in humans today is an indisputable fact. The unique properties of stem cells make it possible to use them in a variety of pathological processes, at different stages and to treat intractable diseases. Stem cell transplantation treats osteoporosis (RF Patent No. 2265442, RU), allergic diseases (Patent No. 2250773, RU), parkinsonism (Patent No. 2259836, RU), brain injury (RF Patent No. 2216336, RU), oncological diseases (Patent No. 2257219, RU) and other diseases.

Currently, a number of serious world veterinary clinics are successfully using similar technologies in the treatment of diseases in animals. The most common problems are arthrosis in dogs, traumatic and degenerative diseases of the spinal cord, which cause paralysis in dogs and cats.

The problem solved by this invention is to create a drug based on stem cells for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals with an expanded spectrum of its use in treatment, as well as to realize the potential of the drug to restore the structure of lost organs and tissues of domestic and farm animals, stimulating their productivity.

The technical result consists in expanding the group of stem cell-based preparations for the prophylaxis and treatment of infectious and non-communicable diseases of domestic and farm animals, as well as in significantly stimulating the milk productivity of cattle, and introducing opportunities and methods for reconstructive cell therapy in pathology of animal organs and tissues, arising from infectious and other diseases.

The object of the invention is solved by the fact that in the method for producing a preparation based on stem cells isolated from pig spleen tissue for the prevention and treatment of infectious and non-contagious diseases of domestic and farm animals, which includes an aqueous solution of polyethylene glycol, an aqueous solution of polyvinylpyrrolidone, a commercial solution Versene, tetracycline solution and commercial trypsin solution in the following ratio of components, vol.%:

15-20% aqueous solution of polyethylene glycol (MM 2000-6000). (PEG) 10-15 15-20% aqueous solution of polyvinylpyrrolidone (MM 280000-360000), (PVP) 10-20 0.01-0.02% Versen commercial solution 10-15 8-10% tetracycline aqueous solution 10-15 0.20-0.25% commercial trypsin solution (100-128 PIECES) Rest

the known composition is introduced into the cell culture medium obtained from pig spleen tissue. The crushed spleen tissue is subjected to antiseptic treatment with a 0.5% solution of metronidazole and subsequent treatment with a 0.05% solution of chlorhexine, followed by disaggregation of the spleen tissue with a 0.25% trypsin solution and cultivation of cell material with a final cell concentration of 1.5⋅ June ¹⁰ -2,0⋅10 ⁶ to 1 ml growth medium Eagle MEM supplemented with 10-15% fetal bovine serum in the presence of a growth stimulator cell culture Pineksida consisting of pituitrina 9 parts and 1 part DMSO. The ratio of components: a known drug and cell-culture medium 5-5.5: 1.

During the development of a method for producing a preparation based on stem cells for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals, the following studies were conducted:

1) the source of stem cells, which plays a role in the subsequent use of cells in the treatment of patients ("Transplantation of hematopoietic stem cells", A. G. Rumyantsev, A. A. Maschan. - Medical News Agency. M. - 2003 - 912 from.). Different sources of MSCs also imply different processing of the initial biomaterial. The conditions for the isolation of MSCs for further cultivation and the conditions for the production of the desired cell mass depend on the composition of the culture medium, the conditions of seeding and cultivation, dishes, etc. The best material for the production of transplant MSCs is fetal material, because cells have a pronounced ability to differentiate, are more active functionally, have great potential for proliferation and growth, produce regeneration and growth factors; when transplanted, they stimulate angiogenesis. The remaining cells better tolerate hypoxia. However, the use of fetal cells is associated with ethical problems and the inaccessibility of the source material.

In the present invention, the spleen of pigs is selected as the source tissue, as it has a greater cell mass for stem cells and is a lethal material in pig production;

2) given that in working with MSCs they use traditional methods of working with cells (Animal cell culture. Methods. Edited by R. Freshney. M .: Mir. 1989), in each case, choosing an individual sequence of actions, conditions for isolation and cell mass production, the present invention has developed a method for isolating stem cells from pig spleen, which includes the following operations:

- mechanical grinding of tissue - the source of stem cells, which is used as a pig spleen, by cutting into small fragments with a volume of 0.2-0.5 mm ³ ,

- antiseptic treatment of crushed spleen tissue by washing on a Shuttel apparatus for 2-4 hours at room temperature in a 0.5% solution of metronidazole in the ratio, mass. 1: 3, respectively, followed by removal of the spent material and washing on a Shuttel apparatus for 6-8 hours at room temperature in a 0.05% chlorhexine solution in the ratio, mass. 1: 3, respectively, followed by the removal of waste material;

- disaggregation of crushed tissue of the spleen by treatment with enzymes - 0.25% trypsin solution 37 ° C in the ratio, mass. 1: 3, on a magnetic stirrer in a thermostat at a temperature of 37 ° C for 15-20 minutes, followed by separation of the supernatant by draining it into a sterile container; this operation is repeated three times, each time draining the supernatant in the same sterile container; at the end of the treatment of the crushed spleen tissue with enzymes, the spent material is removed into waste, and cell material with a cell concentration of 1.5 × 10 ⁶ -2.0 × 10 ⁶ / ml is placed in a sterile culture vessel;

- cultivation of cells from cellular material with a cell concentration of 1.5 × ¹⁰ -2 -2.0 × 10 ⁶ / ml of stem cell growth medium Igla-MEM with the addition of 10-15% fetal bovine serum in the presence of Pinexide cell culture growth stimulator , consisting of 9 parts of pituitrin and 1 part of dimethyl sulfoxide, in the ratio of components, mass. 1: 3: 0.45, respectively; stem cell cultivation is carried out at a temperature of 37 ° C for 48-72 hours in a sterile culture vessel placed in a thermostat.

After 48-72 hours, the cell culture medium is poured into a sterile container. Stem cells can be used for many purposes, in particular for the preparation of a drug for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals. To this end, a well-known drug called “Powin-VM” is introduced into the obtained cell-culture medium (patent No. 2286171, RU, prototype), in the ratio mass. 1: 5-7, respectively, in which the final concentration of the cell-culture medium is 18-20%.

The result is a new drug based on stem cells for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals.

The choice of the concentration of cell-culture medium in 18-20% is due to the following. The low concentration of growth factors secreted by stem cells in the resulting preparation (less than 18%) requires long-term treatment, which is clinically and economically inefficient. A high concentration of growth factors in the preparation (more than 20%) causes allergic reactions at the place of use, focal reactions in the form of swelling, redness, and the formation of edema. General reactions are possible in the form of an increase in body temperature, refusal of food and water, and other signs of clinical depression of physiological functions.

Obtained by the claimed method, a preparation based on stem cells for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals is unique, combining the components of cell fractions, antiseptic compounds, polymer compounds, and has high therapeutic efficacy, confirmed by scientific and clinical studies. The drug is safe to use in the clinic and at work, which is also confirmed by clinical studies. Since the drug is a finished dosage form, it is convenient and easy to use.

Thanks to the addition of the known drug according to US Pat. No. 2286171, RU cell-culture medium, the new drug has much greater efficiency and an expanded spectrum of its application. In addition, the claimed drug has:

- a powerful regenerative potential, manifested in the conditions of the wound process, optimizes the healing of the wound process; stimulates the repair of tendons, ligaments, wound defects, accelerates the restoration of the skin after large area burns;

- osteoinductive properties (accelerating the healing of fractures) and creates the conditions for successful reparative osteogenesis with progressive resorption (reduction) of bone tissue;

- immunostimulating and anti-inflammatory properties, eliminates infectious foci in soft tissues and organs, provides long-term remission and prevention of exacerbations.

The drug has found application in veterinary medicine in its following areas:

1. Obstetrics and gynecology:

- prevention and treatment of mastitis;

- treatment of endometritis;

- stimulation of ovulation reflexes (estrus, hunting in cows);

- increase milk yield in lactating cows;

- treatment of postpartum complications (eclampsia, paresis).

2. Veterinary orthopedics:

- treatment of joint dysplasia;

- treatment of fractures, bruises, bone cracks;

- treatment of ruptures of ligaments and tendovaginitis;

- treatment of bursitis;

- treatment of papillomas on the skin;

- treatment of mycoses.

3. Non-communicable diseases:

- treatment of wounds, trophic ulcers, fistulas;

- stimulation of wound healing in the postoperative period;

- treatment of stomatitis.

4. Infectious diseases:

- viral (infectious) peritonitis in cats;

- leukemia in dogs;

- purulent wounds in animals.

5. Reconstructive veterinary medicine:

- restoration of the anatomical integrity of organs (treatment of monorchism in cats);

- lengthening the stump of the amputated limb in dogs.

The above indicates a wide scope of application of this device and is illustrated by numerous examples of the specific application of the device for the treatment and prevention of infectious and non-infectious diseases of domestic and farm animals, which undoubtedly confirms the expanded spectrum of the use of the created drug in treatment, as well as in the realization of the drug’s potential for restoration of the structure of lost organs and tissues of domestic and farm animals.

The claimed drug expands the group of drugs based on stem cells for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals. This makes it possible to significantly reduce the damage from large waste and weight loss of young farm animals as a result of infectious, viral and many other diseases. The optimal combination of the results of the use of the drug and economic feasibility is the prospect for the introduction of this drug in veterinary medicine.

The invention is illustrated by drawings.

In FIG. 1 is a diagram illustrating the claimed method for producing a stem cell preparation for the prophylaxis and treatment of infectious and non-communicable diseases of domestic and farm animals; in FIG. 2 shows the body weight gain of rabbits injected with the claimed drug per day, compared with the body weight gain of rabbits not injected with the claimed drug per day; in FIG. 3 - the same for California rabbits; in FIG. 4 - FIG. 7 depicts a clinical case of treatment of purulent myositis in a cow in production using the claimed medicinal product (example 23).

The description discloses the best way of carrying out the claimed invention.

An example of a specific implementation of the method of obtaining a preparation based on stem cells for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals.

The drug was obtained and tested in the innovative veterinary clinic of the Tver State Agricultural Academy.

A method of obtaining a preparation based on stem cells for the prevention and treatment of infectious and non-communicable diseases of domestic and farm animals is as follows.

The obtained pig spleen weighing 80 grams is subjected to mechanical grinding by cutting into small fragments with a volume of 0.2-0.5 mm ³ , after which an antiseptic treatment of the crushed spleen tissue is carried out. To do this, the crushed spleen is washed on a Shuttel apparatus for 2-4 hours at room temperature with 240 ml of a 0.5% solution of metronidazole, the spent metronidazole solution is removed by draining and then the subsequent crushing of the crushed spleen is carried out on the Shuttel apparatus for 6- 8 hours at room temperature, 240 ml of a 0.05% chlorhexine solution, followed by its removal. The flushed cell material is treated with an enzyme that weakens or breaks the bonds between cells. The disintegration of the crushed spleen tissue is carried out by treating 240 ml of a 0.25% trypsin solution on a magnetic stirrer in a thermostat at a temperature of 37 ° C for 15-20 minutes

The disaggregation step typically provides a suspension or suspension of aggregated cells and a liquid fraction containing usually free stromal cells (eg, red blood cells, smooth muscle cells, endothelial cells, fibroblasts and stem cells). The next step in the separation method is to separate the aggregated cells from stromal cells.

After processing the crushed spleen tissue with trypsin on a magnetic stirrer under thermostat conditions at a temperature of 37 ° C for 15-20 minutes, remove the supernatant by pouring it into a sterile container; this operation (trypsin disaggregation and discharge of the supernatant) is repeated three times, each time merging the supernatant into the same sterile container.

By the usual methods, for example, using methyl violet dye, the number of nucleated spleen cells in suspension is counted and the growth medium is diluted to a concentration that provides 1.5 × 10 ⁶ -2.0 × 10 ⁶ nucleated cells in 1 ml of growth medium.

As the growth medium, Igla-MEM medium is used with the addition of 10-15% of cattle fetal serum pretreated with ultraviolet rays with a wavelength of 244-264 nm and an emitter power of 30 W for 30-60 minutes; To obtain this serum, it is placed at a distance of 15-20 cm from the radiation source and a thin layer of 0.8-1.0 cm is poured into a sterile Petri dish, after which it is introduced into the stem cell suspension on Igla-MEM medium to a final serum concentration of 10 -15%., And the growth stimulator of cell cultures Pinexid, consisting of 9 parts of pituitrin and 1 part of dimethyl sulfoxide. The ratio of components, mass. 1: 3: 0.45, respectively.

Cultivation is carried out in a culture vessel such as a plastic mat with a capacity of 1.5 liters. The culture vessel is placed in a thermostat and for 48-72 hours at a temperature of 37 ° C carry out the cultivation of stem cells of a pig spleen.

This stem cell isolation technology has a direct connection with the source material, the features of which dictate the choice of the number of operations, parameters and conditions for their implementation.

A well-known preparation called “Powin-VM” is introduced into the obtained cell-culture medium (patent No. 2,286,171, RU, prototype), in the ratio, mass. 1: 5-5.5, respectively, as a result, the final concentration of the cell-culture medium is 18-20%.

The therapeutic and prophylactic effect is confirmed by the results of testing the inventive drug in the clinic and at work, given in examples of specific applications.

Treatment and prevention of a wide range of animal diseases, as well as reconstruction of affected organs and tissues in a short period of time, is carried out in the innovative veterinary clinic of the Tver State Agricultural Academy, in the veterinary clinics of the Tver and Moscow regions, where exclusive methods of rehabilitation therapy using stem cells are used.

Example 1. In October 2012, a cat with clinical signs of monorchism (according to ultrasound data) was admitted to the innovative veterinary clinic of the Tver State Agricultural Agricultural Academy. The drug was administered into the scrotum once every 7 days at a dose of 0.3-0.5 ml per administration. After 8 injections of the drug, a second testis appeared in the scrotum (according to ultrasound).

Example 2. In February 2014, the French Great Dane with a clinic for hip dysplasia was admitted for treatment to the innovative veterinary clinic of the Tver State Agricultural Agricultural Academy. Gait disturbances, lameness, pain, bursitis, tendovaginitis of the left hip and elbow joints were noted. Intradermal chipping was performed along the spine in the region of the hip and elbow joints, periarticular regions at a dose of 0.2-0.4 ml per injection point once every 7-14 days for 4 months. As a result of treatment, lameness and pain stopped.

Example 3. In March 2014, a Sheltie dog (mini Collie) was admitted to the innovative veterinary clinic of the Tverskoy State Agricultural Academy, in which the lower third of the left hind limb was removed. The drug was injected into the stump: 0.2-0.4 ml, 1 time in 7-14 days. After each injection, the stump grew by 1.7-2 cm. Strong cartilaginous tissue was noted on palpation. The dog, previously moving on three legs, began to rely on the grown stump of the left limb.

Example 4. In June 2014, a cat with a clinic of infectious (viral) peritonitis was admitted to the innovative veterinary clinic of the Tver State Agricultural Agricultural Academy. Intradermal administration of the drug along the spine above the liver to the injection point of 0.2-0.3 ml and the abdominal cavity at a dose of 2-3 ml of the drug led to a complete recovery.

Example 5. In April 2014, an East European Shepherd dog with a microsporia clinic was admitted to the innovative veterinary clinic of the Tver State Agricultural Agricultural Academy. The lesion was injected intradermally with a dose of 0.2-0.4 ml per injection point once or several times. Rapid restoration of the focus was noted: the crust disappeared, hair growth appeared.

Example 6. In May 2013, a toy terrier dog with chest deformity was admitted to the innovative veterinary clinic of the Tver State Agricultural Agricultural Academy.

Indications for treatment:

- pain in the chest;

- two papillomas (one on the back right paw, the other on the back);

There was also no front incisor (it fell at the age of 2 years).

Treatment:

The course of therapy with the proposed drug intradermally at a dose of 0.2 ml per injection point 1 time in 7 days for 1.5 months along the spine.

Results:

- The pain in the sternum decreased, and after 2 months the dog did not experience pain when pressing the sternum.

- papillomas disappeared a month after the injection.

Six months after treatment, a new tooth unexpectedly grew.

The results of the use of the drug for the treatment of mastitis in cows and immune stimulation of young animals in Orekhovo-Zuevo-Milk LLC, Orekhovo-Zuevsky District, Moscow Region.

Example 7. In March 2013, the drug was tested in LLC Orekhovo-Zuevo-milk, Orekhovo-Zuevsky district of the Moscow region. Total processed 40 goals. Discharged from the mastitis group after the full recovery of 22 heads. Of them:

- after a single use of the drug - 12 goals;

- after double use - 7 goals;

- after triple use - 2 heads;

- after four times use - 1 head;

- on launch - 1 head.

Relapse - 1 head. Eliminated - 2 heads.

Example 8. In March 2013, the drug was tested in LLC Orekhovo-Zuevo-milk, Orekhovo-Zuevsky district of the Moscow region. A total of 74 heads were processed. Discharged from the mastitis group after full recovery - 37 goals. Of them:

- after a single use - 17 goals;

- after double use - 12 goals;

- after triple use - 4 heads;

- after four times use - 2 heads;

- launch - 2 heads

Relapse - 3 heads. Eliminated - 1 head

This drug was treated not only with acute forms of mastitis, but also “chronicles”. The best results were obtained from cows with increased somatic, blood in milk and catarrhal forms of mastitis. Cases were observed when the full recovery of the cow occurred 3-4 weeks after the last use of the drug without any additional treatment.

Example 9. Prevention of diseases of calves of the Orekhovo-Zuevsky district of the Moscow region. In April 2013, the drug was tested in Orekhovo-Zuevo-Milk LLC of the Orekhovo-Zuevsky District of the Moscow Region. We introduced the drug on the second day of the calf’s life subcutaneously in the preproduction area at a dose of 1 ml. Before using the drug, our main problem was calf gastroenteritis in the calf after being transferred from the maternity ward. After the start of use, the number of calves with gastroenteritis disease was halved, the disease was tolerated much easier and the course of treatment was reduced by 1-2 days.

The results of the use of the drug on rabbits in Bezh-Miakro LLC, Bezhetsky district, Tver region.

Example 10. In September 2014, the drug was tested in Bezh-Miakro LLC, Bezhetsky district of the Tver region. All rabbits were in the same conditions. Taking into account the breed, gender, age, body weight, 16 rabbits were selected, of which 2 groups were formed. The first group is control, the second group is experimental. In the experimental group were rabbits patients with a diagnosis of Rhinitis, Conjunctivitis. The rabbits of the second group were injected intramuscularly, in the back of the thigh, at a dose of 0.2-0.5 ml - once. The drug was not administered to rabbits of the control group, vaccinations were carried out against myxomatosis and viral hemorrhagic disease of rabbits, during which time they were treated against eimeriosis according to the plan of antiepizootic measures, despite the fact that the rabbits were treated twice with Tolucox 2.5%, according to the instructions, clinical signs of eimeriosis appeared. Diseases and deaths among rabbits injected with the claimed preparation were not noted. During the first week, the clinical signs of rhinitis and conjunctivitis stopped.

Thus, we can conclude that the proposed drug:

- increases the safety of rabbits after weaning 100%.

- increases the gain in live weight.

- Prevents diseases of the gastrointestinal tract of rabbits during weaning and when changing feed.

- increases the resistance of rabbits to the negative impact of adverse stress factors,

- activates the growth and development of rabbits.

- non-specific prophylaxis and treatment of infectious and invasive diseases.

- stops the inflammatory process.

The inventive preparation is simple to use, has no side effects, does not require additional use of antimicrobial and anti-inflammatory drugs and coccidiostatics and will be appropriate in combination with specific prophylaxis against HBV and rabbit myxomatosis.

For this drug, a future perspective for environmentally friendly rabbit meat. Newborn rabbits grow very quickly. The average live weight of 4.7-5.2 kg is a rabbit, 4.5-5 kg is a rabbit (Fig. 2 and Fig. 3).

The results of the use of the drug in the Kashin station to combat animal diseases.

Example 11. In May 2014 in the village of "Buzykovo", LPH Grafova K.N. the drug was tested in the pathology of the reproductive organs of the mammary gland. Cow "Rose" age 12 years old №32. The animal has good milk productivity, however, it came to the hunt and successfully covered once a year, the last calving was in the spring of 2013. Accordingly, the lactation period was not 300 days, but about two years. During this time, milk productivity declined, but remained at an acceptable level for owners. In May 29 and June 5, 2014, the drug under study was administered twice, at five points (the occipital region was one injection and 2 injections in the prepopular and lumbar regions, retreating 2 cm from the spinous processes). The introduction was carried out subcutaneously since inside, skin administration in a dose of 0.5 ml at five points is painful enough for a cow and, accordingly, is wary of owners of animals. The animal came into the hunt 5 days after the first injection. Prior to this, signs of hunting were noted 6 months ago. The daily milk yield on the date of the first administration was 18 liters. After 3 days, the volume of milk began to increase and a week after the second injection reached 25-26 liters. Against 18 on the day of the first injection. Milk productivity remained at a high level until mid-September.

Example 12. In August 2014 in the village of "Pervomaiskoe", Kruuz I.V. the drug was tested for pathology of the udder and reproductive organs. Cow "Katya" No. 75, age 14, milk yield 6 liters. In the spring, appetite deterioration, gait shakiness were noted, the animal became more difficult to leash, because she walked slowly to the field with stops. The drug was introduced 08.08.14, at five points once, subcutaneously. After 7 days, signs of hunting were noticed. The animal was led to a bull, but fertilization did not occur after covering. Then, within a week, an improvement in appetite was noted, the gait recovered, the animal began to “frolic,” which had long been gone. Milk productivity did not increase, but regular ovulation recovered (1 time in 3 weeks), until this last time signs of hunting were noticed in the spring. The animal began to gain weight. At present, owners do not have any deterioration in cow health.

Conclusion: In the above cases, there is an obvious connection between the administration of the drug and the restoration of ovulation in animals, an improvement in the general physiological state, an increase in milk production, or, as in the second case, the cessation of weight loss and the restoration of the former fatness of the cow. Thus, the obtained result suggests the possibility of effective use of the claimed drug to extend the life of dairy cows and to improve the pre-slaughter feeding of culled animals. The effect obtained after the use of the preparation by a 14-year-old, very elderly cow suggests a very good restoring effect of this drug in younger animals that have had a serious illness (in practice, treatment is carried out comprehensively, which makes it difficult to give priority as any drug.

The results of the use of the tested drug in the Toropetsk station to combat animal diseases.

Example 13. In April 2014, a dog of the breed Laika with a lacerated wound on the body entered the Toropetsk SBBZH. A course of treatment of the dog with the claimed drug was conducted in the period from April 6 to April 19, 2014.

During a clinical examination at the initial visit, it was found that a skin-muscle flap was torn off of a dog in the area of the right forearm, which was held on a piece of muscle skin about 15 mm wide. The wound is heavily contaminated, there are areas of necrosis, and an odorous smell. The wound was received 7-10 days ago. All this time the dog was not at home (ran away during the hunt). During surgery, the muscles and skin were cleaned, necrotic areas were removed, and the torn flap was connected to the forearm tissues using sutures. Immediately after suturing, the dog was injected with this drug at a dose of 3 ml subcutaneously in the region of the right shoulder blade.

Five days later, when examining the wound, it was noticeable that on the lateral side the suture was severely inflamed, the wound was as if opened, although the sutures were preserved. Serous exudate with fibrin elements is released from the wound. The sutures were treated with antiseptic solutions and made a second injection of the studied drug in the same dosage. Five days later, complete healing, restoration of innervation and blood supply were revealed. After a few days, the restored flap did not stand out above the surface of the paw, there were no scars left. The dog is active and already accompanies the owner on the hunt.

Treatment with the claimed drug helps to restore the affected tissue more fully and in a shorter time.

Example 14. In April 2014, a dog of the Central Asian Shepherd breed with a left forearm injury, with an alleged fracture of the humerus, was admitted to the Toropetsk SBBZh. A course of treatment of the dog with the drug was conducted in the period from April 26.-May 10, 2014.

During the clinical examination at the initial visit, it was found that the dog was taken from the shelter, and from there they gave information about the injury to the left forearm (possibly a fracture). Whether or not it was treated is unknown. At the time of examination, the forearm has severe swelling, purulent exudate is released, the animal has a fever. On the x-ray - ostitis. The exudate was evacuated, the skin in the edema area was treated with an antiseptic solution. This drug was injected into the area of the alleged injury, subcutaneously, in a dose of 3 ml.

Seven days later, upon examination, it was noticeable that the swelling was asleep. The temperature is within the physiological norm. Edema occurred in the shoulder joint, but, according to the owner, lincomycin was injected into this area, and subcutaneously. Perhaps swelling in the shoulder joint is a consequence of this. They made a second injection of this drug in the same dosage, lincomycin was canceled. Seven days later, the dog came without swelling, limping slightly. The test drug was no longer used. On subsequent visits, lameness remains.

Treatment with an approved drug stops the inflammatory process in deeply lying tissues with the consequences of chronic injuries.

Example 15. In April 2014, a thoroughbred cat with a lacerated infected wound in the neck entered the Toropetsk SBBJ. A course of treatment of the cat with the drug was carried out in the period from April 18 to May 16, 2014

A clinical examination during the initial visit revealed a lacerated infected wound in the neck, applied about 10 days ago. At the time of inspection, the edges of the wound are dead, there are overlays of fibrin, there is no skin flap to close the wound. The area of the wound is 3 × 3 cm. The skin around the wound is thickened, inelastic. The edges of the wound were sutured. The test drug was injected intradermally, at a dose of 0.3 ml at one point, there are 4 such points.

An attempt to tighten the edges of the wound was unsuccessful, the seams parted completely after 4 days. But at the same time, the skin acquired a more lively appearance, became more elastic, the area of the wound decreased. Spent another injection of the claimed drug according to the same scheme as the first time. Seven days after examination, the skin is pink, warm, elastic, the edges of the wound come closer. The skin is clearly growing. The muscles on the surface of the wound turned pink, fibrin overlays disappeared. This drug is no longer used. At subsequent examinations, the wound was closed without complications, and wool growth began.

Treatment with the test drug accelerates the regeneration of damaged tissues.

Example 16. In August 2014, a Persian breed cat with a clinic of demodicosis and ringworm was admitted to Toropetskaya SBBZH. The cat was treated with the claimed drug in the period from 08.21.-08.28.2014.

At the clinical examination, at the initial visit, demodecosis and ringworm were diagnosed. At the time of examination - skin manifestations of demodicosis, the coat is rare, greasy, yellowish (natural color - pure white). Conducted treatment with the drug "Wackderm". No positive result was found. With subsequent administration under the skin area affected by ringworm, this drug was administered in a dose of 0.5 ml.

When taken after 7 days, it was revealed that the manifestations on the skin sharply decreased both qualitatively and quantitatively. At the same time (the cat is long-haired and with a white color), the coat has acquired a bright white color and has become silky to the touch.

After a week, treatment of the animal was discontinued due to complete recovery.

The inventive preparation has a positive effect on the quality of the skin and coat.

Example 17. In December 2014, a thoroughbred dog with hind limb paresis was admitted to Toropetskaya SBBZH. A course of treatment of the dog with the drug was conducted in the period December 14, 2014 - April 2, 2015

According to the owner, the dog has been ill for about 3 months. Paresis of the hind limbs developed. At the time of inspection - a shaky gait, fingers tuck back when walking, the tail completely lost sensitivity. The animal often falls when moving. Subcutaneously in the area of both hip joints, this drug was administered in a dose of 0.5 ml.

When taken after 7 days, it was revealed that the dog began to rise better, his gait became more confident, he could wag his tail, but he could not lift. Conducted a second injection of the study drug in the same way. With subsequent administration, an improvement in locomotor activity is noticeable and has better tail control. Every seven days for several months, injections with this drug were carried out according to the same scheme (at the request of the owner). Full recovery did not come. In March 2015, the hostess refused to continue treatment and the dog returned to its original state (paresis of the hind limbs and tail) within 5-7 days.

Example 18. In April 2015, a thoroughbred cat with a fragmented paw entered the Toropetsk SBBZH. A course of treatment of the cat with the drug was carried out in the period 04/05/2015 - 04/04/2015

According to the owner, it was found that the cat is a street cat, picked up on the street with a fragmented paw. The paw is amputated 3 cm below the elbow joint. Healing went unsatisfactory, there was a constant, quite plentiful exudation. After 5 days, the stitches parted, the bone was exposed. We performed a second operation, amputated paw along the elbow joint. At the end of the operation, 0.5 ml of the studied drug was injected intradermally in the stump area. Healing went very quickly, without complications. On the seventh day the stitches were removed, the scar is almost invisible.

The use of the claimed drug provides good tissue regeneration.

Example 19. In April 2015, a thoroughbred cat with no hair on the body entered the Toropetsk SBBZH. The cat was treated with the claimed drug in the period from April 18-28, 2015.

According to the owner, it was found that the cat bald against the background of the stress received in connection with the change of residence and owner.

Clinical examination revealed a complete absence of hair on the body of the animal. The hair is present on the head, neck, tail, but it is rare, stiff.

The drug was applied at a dose of 0.5 ml subcutaneously, at five points (in the occipital tubercle, to the left and right of the spinal column in the region of the shoulder blades and sacrum).

After five days, the cat's well-being improved, the hair began to grow back. The melon preparation injection course was repeated two more times. The quality of the coat has improved. The animal was covered with short hair.

The inventive drug has a beneficial effect on metabolic processes, as well as on the nervous system.

Example 20. In June 2015, cats with combined body trauma were admitted to Toropetsk SBJ. A course of treatment of cats with the drug was conducted during the period from June 8-18, 2015

With an interval of two days, two cats with almost the same injuries entered the veterinary clinic.

The first was a purebred cat. Pregnancy was 2 months. The cat was found beaten, a strange form of the abdomen, does not get up, the temperature is 39.7 ° C. On examination revealed - rupture of the abdominal wall, prolapse of the pregnant uterus under the skin. Laparotomy revealed multiple uterine ruptures, dead fetuses in the abdominal cavity, only one of them in the fetal bladder, peritonitis, hematomas of the abdominal wall, subcutaneous tissue, internal fat, a significant amount of transparent red exudate in the abdominal cavity. The uterus was amputated, the fetus was evacuated, the exudate was evacuated, and necrotic sites were removed.

The second was a purebred cat. Received with a preliminary diagnosis of traumatic hernia. Diagnostic laparotomy revealed tears of the abdominal wall in three places, the size of the tears from 4 to 10 cm. The intestines, bladder, kidneys, spleen fell under the skin. Both kidneys are chicken-sized, painless, cherry bladder. During the operation, the organs were corrected, the gaps were closed.

After the operation, the studied drug was used for both animals. To a cat - in a dose of 1 ml on both sides of a seam of 0.5 ml .; cat - 1.5 ml subcutaneously at two points in the kidney area. After 7 days, the animals were given for examination, healing is going well, the animals are active, the temperature is within the physiological norm, they are willing to take food. This drug was no longer used, the sutures were removed on the 10th day.

The use of the studied drug provides good tissue regeneration and has a beneficial effect on the immune system and metabolic processes.

Example 21. In May 2015, a thoroughbred horse with edema in the withers and croup of an unclear etiology arrived in Toropetskaya SBBZH. A course of treatment of the horse with the claimed drug was carried out in the period from 05.15.-01.07.2015

According to the owner, the horse became ill about three weeks ago. On the neck under the mane, in the area of the withers and croup, there were test-like painless swelling, a wooden gait.

Clinical examination revealed a lack of appetite, a wooden gait, unwillingness to move, head lowered, temperature, pulse, breathing within physiology. On palpation, a slight soreness in the kidney area is detected.

An approved drug was used at a dose of 5 ml subcutaneously in the kidney area. No improvements have occurred. After a few days, the animal stopped rising, symptomatic treatment failed. The horse was lying on its side and refused to eat and drink. This drug was introduced, which was instilled into the animal’s eyes in a dose of 5 drops. The horse began to hold his head, lies on his stomach and willingly accepts food and water. For several days, the horse, with all the positive dynamics, did not stand on the limbs. The observation was carried out for some more time, but the horse had to be euthanized.

The results of the use of the drug in JSC "Agrofirm Dmitrova Gora."

Example 22. From January to September 2015, production and economic testing of the claimed drug was carried out on newborn calves in the amount of 200 goals. The drug was administered to calves of three days old subcutaneously in the lumbar and thoracic spine on the right side at a distance of 5-7 cm from the spinal column, at a dose of 2-4 ml per head, once. The control group of calves was not treated with this drug.

On the 7th day, the control and experimental groups had diarrhea, however, in the calves treated with the above preparation, the feces had a small volume without a specific acid smell. The control group showed excretion of feces in a very large amount and with an acidic odor.

After 2 months, the experimental and control group of calves was transferred to feedlots. A week later, in the control group of calves, an oppressed state, refusal of food, coughing and other signs of pasteurellosis were noted, and in the group treated with the proposed drug, the above clinic was noted after 3 weeks and proceeded in a milder form.

This drug has also been used for inflammation of the umbilical cord in newborn calves. The treatment results showed a 100% recovery.

With purulent myositis, only 2 injections of the claimed drug led to a 100% cure after 20 days, instead of 60 days in the control group (see illustrations at the end of the act in the dynamics of treatment).

As a result of studies of the studied drug on calves and cows, it was noted:

1) Treatment of calves with a drug at the age of 3 days led to the termination of the clinic of pasteurellosis;

2) The proposed drug effectively suppresses the inflammatory process in purulent diseases of calves and cows.

Example 23. In January 2015, the shelter suffering from purulent myositis was treated with the claimed drug. As a result of trauma to contaminated objects, purulent myositis occurs in cows, which cannot be treated in animal husbandry. The reason for this is prolonged antibiotic therapy and low treatment effectiveness. The claimed drug was tested for the treatment of this pathology. We present the results of treatment with the studied drug of one of the cows suffering from purulent myositis.

As a result of purulent fusion of muscle tissue in the right thigh region of the cow, a cavity was formed into which the human brush penetrated (Fig. 4).

As a result of muscle chipping with the studied drug, the cavity began to decrease after 5-7 days, purulent discharge ceased, new muscle-skin elements began to form. The dynamics of the cow's movement improved and the limp of the leaning limb stopped (Fig. 5).

After 5-6 days, the cavity was intensely tightened, a dense scar was formed from muscle elements and connective tissue (Fig 6).

The result of treatment with the studied drug on the 20th day showed: a dense scar formed, the general condition of the cow is good, there is no lameness (Fig. 7).

Thus, the use of the test agent allows you to restore the functional activity of the limb by eliminating purulent cavities in a significantly short period of time.

The results of the use of the drug in the Kimra station to combat animal diseases.

Example 24. This drug began to be used since December 2014. We present several clinical cases in different animals with different diseases using the claimed therapeutic agent. A dog of the Central Asian Shepherd breed suffered from a tumor in the region of the left shoulder blade. The tumor size is 35 × 25 cm, the dog does not interfere with moving. The tumor formed 2.5 months ago. At the three points of the tumor, the inventive preparation was administered, 2.5 ml per administration. After 10 days, the palpation of the tumor was soft and the skin drooped in several places due to the reduction of the tumor, painless. After 16 days, the tumor was completely absent, and the proposed drug was introduced into the resulting skin pockets. After 25 days, the tumor completely disappeared.

Example 25. In January 2015, a cat was hit by a car. For treatment, the proposed agent was applied to the nape of the neck at three points of 0.2 ml per injection, then 2 injections into the spine behind the shoulder blades. On examination, it was noted that the cat had closed fractures of the front and rear limbs on the right side, urine with blood. Every 3 days, the claimed drug was used at the same injection sites. After 14 days, the cat got to his feet, and after another 4 weeks he began to move on the surface without visible signs of injury. Gypsum dressings were not applied except bandaging the injured limbs.

Example 26. In February 2015, we were approached with a sick guinea pig at the age of 2 years with signs of refusal to feed, complete immobility, weak heartbeat. The proposed agent was applied to the spine behind the shoulder blades. The animal quickly returned to normal, after 2 days the procedure was repeated.

Example 27. In March 2015, a cat was admitted with extensive mastocytoma at the age of 3.5 years. Due to the weakness of the animal, the operation was abandoned. Used the claimed drug in a volume of 5 ml once under the tumor and directly into it. After four times the use of this drug, the tumor decreased in half, and after the 8th injection, the tumors completely disappeared.

Example 28. The proposed drug was tested in April 2015 in the SEC "Ilinskoe" for the treatment of calves suffering from severe dyspepsia. On examination: severe calf weakness, signs of dehydration, copious amounts of yellow liquid feces, mixed type of shortness of breath, refusal to feed and milk. Against the background of rehydration therapy, the claimed preparation was applied subcutaneously at a dose of 2-3 ml per head in the interscapular and sacral region. After 2 days, the injections of the test drug were repeated. After the first injection, the lying calves began to rise, take milk and food, and after 3 days the diarrhea completely stopped, the calves recovered.

The claimed drug has a number of advantageous qualities. Stem cells introduced into the wound secrete the main fibroblast growth factor that affects accelerated angiogenesis. This process in the tissues leads to the fact that the cells accompanying the newly formed vessels (pericytes), which preserve the potential of stem cells, are stem cells (pluripotent) and are able to differentiate into fibroblasts and osteoblasts. Regenerative osteogenesis and histogenesis are carried out by stimulation of osteogenic and other cells, with the mandatory participation of growing, newly formed blood vessels.

The obtained positive results of the use of the claimed drug in an animal experiment made it possible to use it for plasty of affected bone and soft tissues, as well as organs, including skin and derivatives.

The introduction of the developed drug to young and adult livestock of farm and domestic animals and the engraftment of stem cells among pathologically altered cells of this organism creates a unique situation: stimulating development and renewal factors that are characteristic only of the embryonic period begin to act on the cells and organs of an adult or young organism. This drug is able to optimize the processes of regeneration of bone and soft tissues, or to contribute to the healing process of affected organs and tissues. The main growth factor of spleen stem cells positively affects the development of all types of cells in the wound, affected bone tissue, and ligaments of the joints. Transforming growth factor stimulate chemotaxis in the affected foci, stimulate angiogenesis and canalization of regenerating tissues. These growth factors can accelerate the recovery of the affected dermis in various skin diseases, damage to the eyes, internal organs, healing of fractures, restoration of the functions of internal organs, etc.

The claimed drug is tested from 2012 to the present with a positive result: it is a safe and highly effective drug for the treatment of many infectious and non-communicable diseases of animals. In particular, this drug contributes to the rapid treatment of diseases such as microsporia of cats and dogs in a few days after a single use, in case of bone fractures, chipping of the focus leads to the appearance of bone marrow and restoration of bone tissue in 10-15 days or even less. In a few days, this drug is able to eliminate the clinical manifestations of a number of eye diseases (corneal inflammation, clouding of the lens, inflammation of the eyeball and conjunctiva), in case of rhinitis, otitis media, otitis media, dermatitis, peritonitis, hepatitis, ascites, the studied drug can significantly improve the quality of life animal in severe forms of the disease, and in most cases lead to complete recovery. All of the above makes it possible for its widespread adoption in the veterinary clinic and practice.

Please assign the name “Theocomposite” to the proposed drug, according to the approved documentation (Makaryan, E.A. Theocomposite - stem cell-based biotechnologies in reproductive cell technology: Guidelines / E.A. Makaryan. - Tver, Tverskaya State Agricultural Academy, 2014. - 15 from.).

Claims (5)

A method of obtaining a preparation based on stem cells isolated from pig spleen tissue for the prevention and treatment of infectious and non-infectious diseases of domestic and farm animals, which includes an aqueous solution of polyethylene glycol, an aqueous solution of polyvinylpyrrolidone, a commercial solution of Versen, a solution of tetracycline and a commercial solution of trypsin with the following ratio of components, vol ./%: 15-20% aqueous solution of polyethylene glycol (MM 2000-6000), (PEG) 10-15 15-20% aqueous solution of polyvinylpyrrolidone (MM 280000-360000), (PVP) 10-20 0.01-0.02% Versen commercial solution 10-15 8-10% tetracycline aqueous solution 10-15 0.20-0.25% commercial trypsin solution (100-128 PIECES) Rest characterized in that the known composition is introduced into the cell culture medium with a final cell concentration of 1.5 × 10 ⁶ -2.0 × 10 ⁶ / ml, obtained from the crushed tissue of pig spleen treated with 0.5% solution of metronidazole and 0 , A 05% solution of chlorhexine followed by its disaggregation with a 0.25% trypsin solution and cultivation of cell material in Igla-MEM growth medium with the addition of 10-15% fetal bovine serum in the presence of Pinexide cell culture growth stimulator, in the ratio mass. 5-5.5: 1.

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