RU2412993C2 - Method of obtaining blood with presence of hematopoetic and stromal stem cells from fetus-calves - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: pregnant uterus is extracted after visceration of incalvers, fetus is freed from child and maternal placentas without injuring and amputation of umbilical cord. After that blood is sampled from jugular vein of 4-5 month fetuses of calves. Vacuum space in thoracic and abdominal cavities of fetus, which prevents free flowing out of blood (hydrodynamics) is eliminated by method of their puncture with trocar, antioxidant is added to blood, settled, centrifuged.

EFFECT: method makes it possible to obtain larger amount of blood rich with hematopoetic, stromal stem cells, enzymes and nucleus-containing regular elements of blood.

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Description

The invention relates to the field of veterinary medicine, namely to cell technology in veterinary medicine.

There is a method of taking blood from rabbits, where blood is taken with a syringe from the jugular vein or from the heart by piercing it with a needle or a tip of a scalpel (1).

However, this method is unsuitable for taking blood from newborns or fetuses in order to collect blood having bactericidal enzymes, stem stromal cells. Taking blood from the ear vein of a rabbit with a needle is complicated by the low speed of blood flow, because periodically, the walls of the blood vessels of the capillaries subside, it is necessary to massage the auricle in several steps, having received blood vessels filling, one by one collecting blood into a syringe and piercing the wall of the blood vessels of the auricle of the rabbit a few times with a blood-pricking needle.

Also known is a method of taking blood from nutria, when the animal is fixed, the limbs are heated in hot water to 50 ° C and one of the limbs is opened where the blood vessels pass, to take blood. Take blood in a test tube with a 5% solution of sodium citrate in a ratio of 1:10 (2).

The disadvantage of this method is that although this method is needleless and there is no spasm of the blood vessel walls, periodic heating of the extremities, cutting off claws with the capture of soft tissue of the hind paw creates stress factors of a pain nature. An open connection with the environment when taking blood from nutrias does not ensure its sterility. When the temperature decreases when the limbs are heated, the blood flow decreases or stops, so you have to take blood in small doses and in several doses.

As the closest analogue to the prototype, a method for producing nucleated cells from the umbilical cord (3) was adopted, characterized in that the nucleated cells are collected with umbilical cord blood, which is collected in two stages, in continuous mode by exfusion from the distal umbilical vein with subsequent application of an anticoagulant, and the remaining blood is collected by perfusion. Blood collection is carried out in two containers, stirred alternately, the supernatant is separated, centrifuged at 2000 rpm, 10-12% dimethyl sulfaxide in polyglucin is added, followed by freezing and further therapeutic use.

However, this known method is unsuitable for obtaining fetal blood rich in nucleated cells. The disadvantage of this method is to obtain a small amount of cord blood. This is due to a change in pressure in the thoracic and abdominal cavity of the fetus, when the blood vessels are running, therefore, it is necessary to collect blood from the umbilical cord in several stages, using exfusion, perfusion, receiving cord blood in small doses, using several containers with the addition of anticoagulant to collect blood. The process of collecting blood is laborious.

The technical solution to the problem is to increase the efficiency of taking blood from cattle fruits by canceling the process of spasm of the walls of blood vessels and blood coagulation in the blood vessels of the umbilical cord.

The problem is achieved by the fact that the method of taking blood from 4-5-month-old calf fetuses taken from pregnant cows slaughtered as a result of declaring cattle farms and collective farms bankrupt is used.

When performing the method, the procedures were performed in the following order:

- removal of the pregnant uterus after the nesting of pregnant cows;

- release of the fetus from the infant and maternal placenta;

- taking blood from the jugular vein of the fetus;

- adding to the blood an antioxidant, sedimentation, centrifugation, characterized in that:

- the fetus is released from the infant and maternal placenta without injury and amputation of the umbilical cord;

- blood is taken from the jugular vein from 4-5-month-old calf fetuses;

- the vacuum space in the chest and abdominal cavity of the fetus, which prevents the free flow of blood (hydrodynamics), is eliminated by puncturing them with a trocar.

The ongoing manipulations in the proposed method lead to the maximum obtaining the amount of blood necessary for collecting blood, and also does not increase the risk of microbial infection. The blood collection process takes place without contact with the mother, quickly, does not pose any risk to the meat products of slaughtered carcasses of cows received for slaughter from the slaughterhouse in the processing department in the refrigerator, to the consumer.

The number of cows is represented by slaughter as a result of a bankrupt farm and must be examined before contamination for contagious and non-contagious diseases.

The method is as follows.

1. Blood collection is carried out in a quartz room.

2. A pregnant uterus with a fetus is placed on a metal table, disinfected, dousing with a solution of manganese acid potassium 1: 1000.

3. Through a longitudinal section of the wall of the uterus of the large horn of the uterus, the fetus is freed from the infant and maternal placentas, from amniotic and allantoic fluids, without injuring the umbilical ring, without amputating the umbilical cord, achieving free visibility of the head, neck, and body of the fetus.

4. Inspect and palpate the jugular gutter, probing the jugular vein in the future from the fetus, which is in a lying position. The external jugular vein on the neck of the fetus is located under the skin in the jugular gutter formed by the brachiocephalic and papillary muscles.

5. With one movement of the hand, a needle bent with a hose (130 °) is inserted into the jugular vein swollen with fetal blood, lying in the jugular groove along the neck of the fetus. To collect blood from the jugular vein, the fetus uses special glass containers or closed transfusion systems containing an anticoagulant such as Gemakon, Baxter, equipped with a sterile drainage needle with a bend of 130 °, corresponding to the topography of the concavity of the jugular trough muscles, which eliminates the obstacle to leakage of blood from the fetus in a supine position.

6. A trocar, leaving the sleeve, removing the stylet, puncture the wall of the abdominal (near the diaphragm), chest cavity, eliminate the vacuum state, which contributes to the unhindered blood transudation through the vessels, through the needle inserted into the jugular vein, the blood flows into the container with the antioxidant.

The presence of a vacuum in the abdominal and chest cavity of the fetus leads to desolation of the blood vessels of the umbilical cord (according to the laws of physiology), so doctors are able to take small volumes of umbilical cord blood from newly born children.

The most abundant formation of shaped elements, nucleated cells, hematopoietic and stromal stem cells, enzymes in fetuses in the blood is achieved in mid-pregnancy, and by the end of fetal development is reduced. Therefore, the most rational is that the blood from fetal calves must be collected at 4-5 months of fetal development.

Practical use

The most important characteristic of fetal blood is the presence in it of quantities of nucleated cells. Samples of the fetal blood were subjected to the method of counting leukocytes in the formula, counting under a light microscope with standard stain smears according to Romanovsky. Studies have shown that in blood samples from calf fruits, there are from 1-0.5 · 10 ⁹ to 2 · 10 ⁹ nucleated cells: leukocytes 7.24 · 10 ⁹ / l to 25.3 · 10 ⁹ / l, platelets from 130 · 10 ⁹ / l to 410.1 · 10 ⁹ / l.

Subsequently, the extracts were fractionated to isolate nucleated cells from the fetal blood by centrifugation of the supernatant at 2000 rpm (20 min). After centrifugation, 10% ointment was made from fractionated fetal blood. Daily, this ointment for 7 days, 100 ml was injected into the cervical canal of cows with postpartum purulent cervicitis (table 1).

Table 1 Treatment of postpartum suppurative cervicitis in cows Method of treatment Sick cows (total) Of them recovered Recovery period (days) number % 5 10 fifteen 10% ointment from fractional fetal blood after centrifugation 12 10 83.3 2 8 2 / 16.7% Furazolidone sticks foaming eleven 6 54.5 3 / 27.2%

After using fractional ointment from fruit blood (table 1) in the form of a 10% ointment for cows in the cervical canal, 83.3% of cows recovering earlier from cervical inflammation recovered on day 10, which recovered 28.8% more cows than after treatment of cows in the control group, to which 5-6 furazolidone foam-forming sticks were introduced into the cervical canal for 7 days. The terms of treatment of the experimental group of cows were 6-10 days less than in the control group of cows.

Bibliography

1. Laboratory research methods in veterinary medicine. M.: State Publishing House of Agricultural Literature. 1954, 223 p.

2. Kataeva T.C. (RU); Lopatin B. (RU). The method of taking blood from nutria. Copyright certificate, patent No. 2236815 C1, Russia.

3. Isaev A.A; Mkheidze D.M .; Grishina V.V .; Prikhodko A.V., patent RU No. 2343928 C1.

Claims (1)

A method of obtaining hematopoietic and stromal stem cells from calf fetuses, which consists in removing a pregnant uterus after calving a pregnant cow, freeing the fetus from the infant and maternal placenta without injuring and amputation of the umbilical cord, taking blood from the jugular vein from 4-5 month old calf fetuses while the vacuum space in the chest and abdominal cavities of the fetus, preventing the free flow of blood (hydrodynamics), is eliminated by puncturing them with a trocar, adding an antioxidant to the blood, settling, centrifuging.