The method of obtaining nucleoprotein complexes from embryos of birds, with immunotropic and radioprotective activity

 

(57) Abstract:

The method of obtaining nucleoprotein complexes from embryos of birds, with immunotropic and radioprotective activity includes laundering and homogenization of the embryos of birds in 0.15 M NaCl, centrifuged in the cold and clean on polyamide hollow fibers with upper limits of selectivity 50 and 5 kDa. table 4.

The invention relates to biotechnology, in particular to the production of medicines from animal products, and may find application in the manufacture of drugs immunostimulating and radioprotective action.

Currently in medical practice used Immunostimulants bacterial origin (prodigiozan, salmozan and so on) [1], and a wide range of products from the thymus (thymosin, timogen, T-activin, and so on) [2 - 4].

The drawback of such drugs is a multi-stage and complexity of the production, use and import of chemicals and equipment, the use of toxic and flammable chemicals. In addition, the bacterial Immunostimulants nature have a wide range of contraindications and side effects (allergic reactions, pain and swelling at the injection site, and so on).

Known methods for producing substances having immunological activity of the embryonic tissues of animals and birds [5 - 8]. However, in all these works were used crude tissue extracts and talk about the application of the results of these works in practice is not necessary.

The analysis of the literature showed that the method of obtaining from embryos of birds of biologically active substances, namely nucleoprotein complexes with immunotropic and radioprotective activity not previously described.

An object of the invention is to develop a method of obtaining nucleotide complexes of the embryos of birds, with immunotropic and radioprotective activity.

The method is as follows. The embryos of birds removed from the shell and washed in 0.15 M NaCl from the blood. To the washed embryos poured equal volume of 0.15 M NaCl and homogenized in razmelchitel tissue for 1 min at a speed of knives 8000 Rev/min, the Homogenate was centrifuged in the cold at 4000g for 15 to 20 minutes, the Supernatant is passed through the hollow fibers of the polyamide with the upper limit of selectivity 5 kDa. Concentrate representing the target fraction, centrifuged to separate denaturirovannykh proteins, and freeze-dried. 100 embryos receive 350 doses of the drug.

In biological terms the target product has a wide range of properties, manifested already at a dose of 0.01 mg/kg of body weight; stimulates phagocytosis and humoral immunity; helps restore immunity, suppressed by chemical means, as well as the recovery of immunoreactivity in experimental animals subjected to x-ray irradiation.

The method is illustrated by the following examples.

Example 1. The introduction of nucleoprotein complexes.

100 eggs with embryos of birds (chickens, quail and so on) received from the incubator, wipe with alcohol, remove the embryos and three times washed with 250 ml of 0.15 M NaCl. To the washed embryos poured equal volume of 0.15 M NaCl (usually 60 to 65 ml) and homogenized in razmelchitel tissue for 1 min at a speed of knives 8000 Rev/min, the Homogenate was centrifuged in the cold at 4000g for 15 - 20 minutes the Supernatant volume 120 - 130 ml is passed through the hollow fibers of a polyamide with an upper limit of selectivity of 50 kDa. To do this, use the installation UPL-0.1 production, Kirishi or similar. On the ml are collected and passed through the hollow fibers of a polyamide with an upper limit of selectivity 5 kDa. Concentrate representing the target fraction in a volume of 25 ml, centrifuged to separate denaturirovannykh proteins, and freeze-dried. To obtain the dosage form, the drug is subjected to sterilizing filtration, bottling according to the dose,and dried by standard methods prescribed for drugs.100 embryos receive 350 doses of the drug immunostimulating radioprotective and anti-tumor activity.

The specific activity of the target fraction is illustrated by the following examples.

Immunostimulirutuyu activity.

Example 2.The study phagocytophilia activity.

Experiments carried out on outbred mice With 57 In 1/6 (males weighing 20-24 g). The target fraction injected intraperitoneally at various doses.At different times after injection conduct research on the status of phagocytic star on nonspecific protection.For this purpose, define the phagocytic activity and phagocytic index.

Data phagocytophilia activity are given in table.1.

From the data table.1 shows that the target faction has a stimulating effect on phagocytosis of peritoneal macrophages in to estimulara activity of the drug.

The influence of the target faction on the humoral immune response estimate simultaneous intraperitoneal administration of the drug and the test antigen (sheep erythrocytes) in a dose of 1 to 107cells/mouse white outbred mice weighing 20 - 25g

Shows the stimulating effect of the target fraction at doses of 0.10 - 10 kg of protein per 1 kg of body weight (table. 2).

Example 4. The study antichlolinesterases activity of the drug in conditions of T-cell immunodeficiency.

The influence of the target faction on humoral immune response in terms of T-cell immunodeficiency appreciate simultaneous intraperitoneal administration of the drug and the test antigen (sheep erythrocytes) in a dose of 1 to 107cells/mouse white outbred mice weighing 20 - 25 g on the background of the preliminary injection of hydrocortisone.

It is shown that the introduction of the target fraction contributes to the restoration of suppressed immunity at its introduction in doses of 0.1 - 10 mg/kg (table. 3).

Example 5. The study of radio-activity.

The influence of the target faction on humoral immune response in immunodepression x-ray radiation dose of 4 Gy experimental animals appreciate simultaneous nutripure the sham weighing 20 - 25,

It is shown that the introduction of the target fraction at a dose of 1 mg/kg contributes to the recovery of immunoreactivity immunodepression x-ray radiation in experimental animals (table.4).

From the description it is seen that the proposed method allows to obtain the target product with a wide range of biological properties, manifested already at a dose of 0.01 mg/kg of body weight:

- stimulates phagocytosis;

- stimulates humoral immunity;

- helps to restore suppressed by chemical immunity;

- helps to restore the immunoreactivity immunodepression x-ray radiation laboratory animals.

These data demonstrate immunocorrective activity of the drug received, suggesting possible areas of application. First, in the treatment of congenital and acquired immunodepressed and immunodeficiencies. Secondly, in therapy of infectious diseases of humans and animals associated with impaired immune status. Thirdly, as an adjuvant during vaccination.

References:

1. Rostunova, A., Shcherbakov, E. G., I. Kruglov, S. Prodigiozan as activator A12 - 817.

3. Low, T. L. K., Goldstein, A. L. Sprinder Semin. Immunopathol., 1979, No. 2, p. 169 - 186.

4. Thurman G. B., Ahmed, A., Strong, D. M. et al. Transplant. Proc., 1975, N 7, p. 299 - 312

5. Polezhaev L. P. Loss and restoration of regenerative capacity of tissues and organs in animals. - M.: 1968, S. 275.

6. Donetsk D. current issues of private surgery. - M.: 1986, S. 173 - 174.

7. Gluhenchi B. Medical business. 1984, No. 6, S. 93 - 94.

8. Axe B. Possible activation of reparative osteogenesis in cancer patients. In the book: VIII national conference of oncologists Moldova. Chisinau, 1989, S. 222 - 223.

The method of obtaining nucleoprotein complexes from embryos of birds, with immunotropic and radioprotective activity, namely, that the money laundering and homogenization of embryos is carried out in 0.15 M NaCl, the homogenate was centrifuged in the cold for 15 - 20 min, the resulting supernatant is passed sequentially through a polyamide hollow fiber with upper limits of selectivity 50 KDa and 5KDa and concentrate representing the target fraction, centrifuged and freeze-dried.

 

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SUBSTANCE: invention relates to isolating and cryopreserving precursor-cells. Methods involve treatment of human liver tissue for preparing the essentially monocellular suspension containing precursor-cells and cells that are not precursor-cells, a single or more lines of cellular differentiation presenting in the human liver. Invention describes methods involving stage for separating cellular population resulting to reducing amount of cells that are not precursor-cells and providing preparing the separated suspension enriched with precursor-cells expressing one or more markers and associated with a single or more lines of the cellular differentiation. Also, invention describes a method for selection cells from the separated suspension wherein these cells or their progeny, or their more matured forms express one or more markers associated with lines of the cellular differentiation. These markers involve: CD14, CD34, CD38, CD45 and ICAM. Hepatic precursor-cells have diameter size 6-16 mc, they are diploid and show indices: glycoforin A-, CD45-, AFP+++, ALB+, ICAM+ and they comprise subpopulations varying with respect to expression of CD14+, CD34++, CD38++ and CD117++. These cells are useful for carrying out cellular and genetic therapy in liver treatment and for preparing artificial organs also.

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