The method of colony-stimulating hematopoietic precursor cells in the spleen when irradiated animals

 

(57) Abstract:

The invention relates to medicine, namely to the stimulants of colony-forming cells. The method is carried out by introducing animals enzymatic hydrolysate of marine life in the amount of 40-50 mg. Stimulating substance is administered to animals before or after irradiation at least 5-7 days. The invention provides a 2-4 times increase the efficiency of colony-hematopoietic precursor cells in the spleen and to accelerate the restoration of blood in animals after exposure. 2 C.p. f-crystals, 1 table.

The invention relates to medicine, namely to the stimulants of colony-forming cells, and may find application in clinical practice and radiobiological experiments.

There is a method of stimulation of colony-forming progenitor cells in the spleen after irradiation by injecting animals with epinephrine in the amount of 0.1-0.07 mg/mouse.

The disadvantage of this method is the low kolonialapologie hematopoietic cells and the use of this pharmaceutical preparation having a specific contraindications for use.

There is a method of facilitating the sa after irradiation Tris(hydroxymethyl)aminomethan in the amount of 0.3-0.5 ml/mouse.

The disadvantage of this method is the use of chemically pure drug, further impact on mammals has not been studied.

The closest in the prior art to the claimed technical solution is the method of action of histone on hematopoietic precursor cells (CFU-c) normal or irradiated body (Radiation biology, Radio-ecology, 1994, 34, No. 4-5, S. 544-549).

The disadvantage of this method is the high cost of the drugs used, which are derived from the thymus of animals by complex biotechnological process.

The objective of the invention is the expansion of supportive funds received from environmentally friendly raw materials, which are able to increase the efficiency of colony-hematopoietic precursor cells in the spleen after irradiation of animals.

The problem is solved by introducing animals stimulating substances, namely enzymatic hydrolysate of marine life in the amount of 40-60 mage. Stimulating substance is administered to animals prior to irradiation, with the last dose of the stimulating substance is administered no later than 1.5 hours before irradiation or after irradiated is TBE stimulant use enzymatic hydrolysates of mussels, or from the mantle scallop or scallop gonads, and the stimulating substance is administered to animals within 5-7 days.

New in the present invention is that as a stimulator of hematopoietic colony-precursor cells in the spleen using fermented hydrolysates of marine life, namely mussels, or from the mantle scallop or scallop gonads, in the amount of 40-50 mg.

Comparison of the proposed method with conventional methods shows that first proposed the use of fermented hydrolysates of marine life: mussels, or from the mantle scallop or scallop gonads in the amount of 40-50 µg, while hydrolysates injected animals daily for 5-7 days before exposure or after exposure, with the last dose of the stimulating substance is administered no later than 1.5 hours before irradiation or the first dose stimulants administered no earlier than 1.5 hours after exposure.

Thus, the claimed invention meets the criteria of the invention of “Novelty” and “Inventive step”.

Enzymatic hydrolysates of marine hydrobionts is prepared as the t separately, add distilled water in the ratio 1:2-1:3, adjusted to pH 8.0-8.2, add standard enzyme proteolytic activity, in particular collagenase, in the amount of 1-5% of the initial mass. The mixture was incubated for 4-8 hours at room temperature, then heated to 100°C., cooled, filtered and dried.

To assess stimulating action of enzymatic hydrolysates of marine life on kolonialapologie hematopoietic precursor cells in the spleen before or after irradiation experimental investigations were carried out on outbred mice weighing 18-20 g

The evaluation of the preventive action of enzyme preparations in experimental animals daily for 5-7 days no later than 1.5 hours before irradiation was injected under the skin in the thigh enzymatic preparations: from mussels, or from the mantle scallop or scallop gonads in the amount of 40-50 µg, pre-dissolve them in 0.85% saline solution. Then mice were irradiated on the device Rocus-M at a dose of 7 Gy at the dose rate of 0.9 G/min

To determine therapeutic action of enzyme preparations animals were first irradiated on the device Rocus-M at a dose of 7 Gy at moderate: mussels, or from the mantle scallop or scallop gonads in the amount of 40-50 µg, pre-dissolve them in 0.85% saline solution, and the first dose of enzymatic drugs were injected animals not later than 1.5 hours after exposure.

On the eighth day after the application of enzyme preparations of mice were scored by decapitation, removed the spleen and within two hours were fixed in liquid Buena. After fixing counted the number of endogenous colonies with the aid of a magnifying glass.

Criterion stimulating effect of enzymatic hydrolysates of marine life is the presence of endogenous colonies like tubercles, size 0.5-3.5 mm, Each colony is a clone of cells arising from a single hematopoietic cells predecessor. The number of colonies in proportion to the number of surviving precursor cells and is an indicator of recovery of blood.

To confirm the stimulatory effect of these funds comparative study was carried out by injecting animals with 40-50 mg of 0.85% saline solution. With the introduction of animal physiological solution in the amount of 40-50 mg before irradiation for 8 days of endocrine number is m by, as the results suggest that smaller doses less than 40 µg stimulating effect of the drug is not significantly different from the control, while increasing dosages above 50 mg the number of colonies is not growing, which indicates the exhaustion of the compensatory capacity and inexpediency of further increasing doses of the drug.

A temporary scheme for the use of the drug is due to pathophysiological laws of development of adaptive reactions of living organisms.

The introduction of the promoter no later than 1.5 hours before or after irradiation provides the necessary stimulation neuroimmunoendocrine system that promotes radioresistance of the body, and the use of stimulants no less than 5-7 days corresponds to the minimum of the traditional regimen of administration of Immunostimulants, providing persistent physiological effect.

Data on the effect of enzyme preparations on the number of endogenous colonies depending on the dose and duration of its application are presented in the table.

The table shows that all taken in the experiment enzymatic hydrolysates from sea organisms are stimulants endogen what's hydrolysates in the amount of 40-50 mg increases the number of endocrine to 1.01 to 2.5 times, moreover, the maximum preventive effect is the use of enzymatic hydrolysate of scallop gonads.

The introduction of irradiated mice enzymatic hydrolysates in the amount of 40-50 mg increases the number of endocrine 1.7-3.9 times, and the maximum therapeutic effect is the use of enzymatic hydrolysate of mussels.

Examples of specific performance

Example 1:

The experiments were carried out on outbred mice-males weighing 18-20 g, which were divided into 4 groups of 10 animals each.

1st group (control) animals were injected with saline.

2nd group - introduced enzymatic hydrolysate of mussels in the amount of 45 g (dry) for 6 days and the last dose for 1.5 hours before irradiation.

group 3 was injected enzymatic hydrolysate from the mantle scallop in the amount of 45 g (dry) for 6 days and the last dose for 1.5 hours before irradiation.

the 4th group was injected enzymatic hydrolysate from the gonads of sea scallops in the amount of 45 g (dry) for 6 days and the last dose for 1.5 hours before irradiation.

Enzymatic hydrolysate of mussels or from the mantle scallop or scallop gonads prasadam mice irradiated at a dose of 7.0 Gy with a dose rate of 0.9 G/min on the phone Rocus) The dose is adjusted in advance empirically and depends on sex, breed and season.

On the 7th day mice slaughtered by decapitation, the spleen removed and fixed in a liquid Buena (saturated solution of picric acid in water - acetic acid - 40% formalin - 15:15:1). After fixation within days of the spleen is transferred into 70% ethanol and count colonies. Counting was done using a magnifying glass.

The table shows that the introduction of enzymatic hydrolysates of marine life increases the number of endogenous colonies on the spleen 1.4-2.5 times, and the use of the hydrolyzate of the gonads scallop gives the highest stimulating effect as prophylaxis, the number of colonies 20,12,3.

The observed increase in the number of endogenous colonies associated with an increase in the number of survivors of hematopoietic precursor cells needed to restore blood.

Example No. 2 was performed similarly to example No. 1, but stimulating substances - enzymatic hydrolysates from marine organisms were injected into mice in the amount of 40 μg(dry) for 5 days.

On the 6th day mice slaughtered by decapitation, the spleen removed and fixed in a liquid Buena (saturated ranki transferred into 70% ethanol and count colonies. Counting was done using a magnifying glass.

The results of the experiment are presented in the table.

The table shows that the introduction of enzymatic hydrolysates of marine life increases the number of endogenous colonies on the spleen in 1.01-1.7 times, and the use of the hydrolyzate of the gonads scallop gives the highest stimulating effect, the number of colonies of 13.5±1,2.

Example No. 3 was carried out analogously to example No. 1, but stimulating substances - enzymatic hydrolysates from marine organisms were injected into mice in the amount of 50 μg (dry) within 7 days.

At 8 days mice slaughtered by decapitation, the spleen removed and fixed in a liquid Buena (saturated solution of picric acid in water - acetic acid - 40% formalin - 15:15:1). After fixation within days of the spleen is transferred into 70% ethanol and count colonies. Counting was done using a magnifying glass.

The results of the experiment are presented in the table.

The table shows that the introduction of enzymatic hydrolysates of marine life increases the number of endogenous colonies on the spleen 1.5-2.5 times, and the use of the hydrolyzate of the gonads scallop gives the most high stocki stimulating effect compared with example No. 1.

Example # 4

The experiments were carried out on outbred mice-males weighing 18-20 g, which were divided into 4 groups of 10 animals each. Then mice irradiated at a dose of 7 Gy with a dose rate of 0.9 Gy/min in the apparatus Rocus) After 1.5 hours after exposure to mice injected stimulating substance. 1st group (control) animals were injected with saline.

Animals of the other groups were injected stimulants in the amount of 45 g(dry) for 6 days. Animals were injected stimulants:

2nd group - enzymatic hydrolysate of mussels;

3rd group - enzymatic hydrolysate from the mantle scallop;

4th group - enzymatic hydrolysate of scallop gonads.

Stimulating substance is dissolved in a 0.85% saline solution and injected under the skin in the thigh once a day, and after the first exposure dose is administered to animals after 1.5 hours.

On the 7th day mice slaughtered by decapitation, the spleen removed and fixed in a liquid Buena (saturated solution of picric acid in water - acetic acid - 40% formalin - 15:15:1). After fixation within days of the spleen is transferred into 70% ethanol and count colonies. On the blitz should the introduction of enzymatic hydrolysates of marine life increases the number of endogenous colonies on the spleen 2.7-3.9 times, and use a hydrolysate of mussels gives the highest therapeutic stimulating effect, the number of colonies and 17.9±5,1.

The observed increase in the number of endogenous colonies associated with an increase in the number of survivors of hematopoietic precursor cells needed to restore blood.

Example No. 5 was carried out analogously to example No. 4, however, the stimulating substance - enzymatic hydrolysates from marine organisms were injected into mice in the amount of 40 μg (dry) for 5 days.

On the 6th day mice slaughtered by decapitation, the spleen removed and fixed in a liquid Buena (saturated solution of picric acid in water - acetic acid - 40% formalin - 15:15:1). After fixation within days of the spleen is transferred into 70% ethanol and count colonies. Counting was done using a magnifier 4x.

The results of the experiment are presented in the table.

The table shows that the introduction of enzymatic hydrolysates of marine life increases the number of endogenous colonies on the spleen 1.7-2.9 times, and the use of hydrolyzed ilogico example No. 4, however, the stimulating substance - enzymatic hydrolysates were injected into mice in the amount of 50 μg (dry) within 7 days.

At 8 days mice slaughtered by decapitation, the spleen removed and fixed in a liquid Buena (saturated solution of picric acid in water - acetic acid - 40% formalin - 15:15:1). After fixation within days of the spleen is transferred into 70% ethanol and count colonies. Counting was done using a magnifier 4x.

The table shows that the introduction of enzymatic hydrolysates of marine life increases the number of endogenous colonies on the spleen 2.4-3.8 times, and use a hydrolysate of mussels gives the highest stimulating effect, the number of colonies at 17.8±4,8.

The observed increase in the number of endogenous colonies associated with an increase in the number of survivors of hematopoietic precursor cells needed to restore hematopoiesis. The introduction of a stimulating substance in quantities of 50 µg gives higher stimulating effect compared with example No. 4.

1. The method of colony-stimulating hematopoietic precursor cells in the spleen in animals that were irradiated by introducing animals stimulating substances,Drobinov in the amount of 40-60 μg, stimulating substance is administered to animals prior to irradiation, and the last dose of stimulating substances injected at least 1.5 h before exposure or after exposure, with the first dose of the stimulating substance is administered not earlier than in 1,5 h after irradiation.

2. The method according to p. 1, characterized in that as a stimulating substance use enzymatic hydrolysate of mussels, or from the mantle scallop or scallop gonads.

3. The method according to p. 1, wherein the stimulating substance is administered to animals within 5-7 days.

 

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