Method of recovering spleen after radiation load

FIELD: medicine.

SUBSTANCE: method is realised by the intravenous allogeneic transplantation of multipotent mesenchymal stromal cells (MMSC) and hematopoietic stem cells (HSC) to laboratory mice one hour after irradiation. HSC are obtained from the placenta of female mice at the 14-day gestation term. MMSC are introduced in a dose of 6.5 mln cells/kg, with HSC being introduced in a dose of 400 thousand cells/kg.

EFFECT: invention makes it possible to extend the arsenal of means, capable of providing the regeneration potential of the spleen tissues, as well as to increase the regeneration of main morphometric spleen indices after exposure to a radiation load.

2 tbl

 

Method of recovery of the spleen after radiation exposure

The invention relates to experimental medicine, namely to develop ways to treat radiation sickness, and can be used to restore the main morphometric parameters of the spleen after exposure to ionizing radiation (radiation load).

Recovery regeneration of spleen is provided by use of drugs that increase the regenerative potential of lymphoid cells. A typical representative of drugs that have a stimulating effect on the regeneration processes is the Derinat (aqueous solution of dezoksiribonukleata sodium). Derinat is a sodium salt of DNA.

The use of this group of drugs is associated with side effects and contraindications. So after the injection of an aqueous solution of dezoksiribonukleata sodium in 1.5-3 h occurs severe hypoglycemia (F. Y. Krylov, E. G. Lobanova. The medicines register No. 12; pp. 303; 2005).

The next step in the treatment of radiation injuries of hematopoiesis is the provision of substitution therapy (bone marrow transplantation) [j. Snell, J. Doss, S. Natanzon // tissue Compatibility. Moscow, MIR, 1979. P. 290-294.].

The disadvantage of this method of therapy is the development of graft-versus-host rejection Tran�of plantat, as well as infection.

The closest is a method of stimulating kolonialapologie hematopoietic progenitor cells in the spleen during irradiation of animals by the introduction of deoxyribonucleic acid extracted from fish ROE. The drug is administered before or after irradiation daily dose of 40-60 µg/mouse for 5-7 days. The last dose of the drug before irradiation or after the first dose of radiation administered over 1-2 hours, respectively, before or after irradiation (RU. Patent No. 2230559, IPC A61K 31/70, AR 7/06, AR 7/00, publ. 20.06.2004).

This method provides kolonialapologie hematopoietic cells in the spleen when addressing side effects, however it is not efficient enough to restore the spleen, since the introduction of DNA not associated with directed migration in the damaged tissue.

The object of this invention is to develop a new technology for restoration of hematopoietic function of the spleen.

The technical result that will be achieved from the use of this invention is to improve hematopoietic function of the spleen.

The technical result is achieved in that in the method of recovery of the spleen after radiation exposure of laboratory mice one hour after irradiation is carried out intravenous allogeneic transplantation of multipotent mesenchymal stromal�tion cells (MSCS) and hematopoietic stem cells (HSCs), obtained from the placenta of female mice during gestation 14 days, MSCS administered in a dose of 6.5 million cells/kg, and GCS in a dose of 400 thousand cells/kg.

The invention consists in the application for the first time to restore the main morphometric parameters of the spleen combined transplantation of cells: MSCS and HSCs isolated from the placenta of female mice during gestation 14 days.

The proportion MMSC (6.5 million cells/kg) and GSK (400 thousand cells/kg) allows you to effectively restore the hematopoietic function of the spleen.

3

From the analysis of scientific-technical and patent literature combined transplantation of cells: MSCS and HSCs isolated from the placenta of female mice during gestation 14 days in the stated proportion, leading to the effective vosstanovlenie hematopoietic function of the spleen, we have not identified that allows to make a conclusion about conformity of the proposed technical solution the criteria of "novelty" and "inventive step".

The invention is as follows.

Materials and methods

General characteristics of laboratory animals used in research

The experiments were performed with 20 white mice-males aged 3-4 months, weighing 20-25 g. the Experiments on the culture of MSCS and HSCs from the placenta performed at laboratory 5 LM�now mice-females age 3-4 months weighing 20-25 g, a gestational age of 14 days. Animals were kept in standard laboratory animal facility, under the "Rules of work with experimental animals" approved by the USSR Ministry of health Order No. 755 from 12.08.1977 G. and the USSR Ministry of health Order No. 1179 from 10.10.1983 "On approval of the standard costs of feed for laboratory animals in health care facilities". Manipulation of experimental animals were performed in accordance with the provisions of the Helsinki Declaration on the humane treatment of animals, methodological recommendations for their elimination from the experience and euthanasia.

On the 7th day, the mice were killed by decapitation, removed the spleen and fixed them in the liquid Buena (saturated solution of picric acid in water - acetic acid - 40% formalin in the ratio 15:15:1). After fixation, during the day the spleen was transferred into a 70% solution of ethanol, and colonies were counted. Counting was performed using an inverted microscope UNICO. The average number of selected endogenous hematopoietic colonies in the application of co-transplantation of MSCS and HSCs was 30.2±1,1, the application of saline - 8,7±1,3.

We studied the influence of ionizing radiation dose of 4.0 Gy on laboratory animals of Mature age, were allocated to experimental and control groups.

Animals on�itoi group was injected intravenously suspension of MSCS and HSCs, respectively, at a dose of 6.5 million cells/kg and 400 thousand cells/kg, control group were injected with 0.9% NaCl solution and 0.2 ml intravenously. Intravenous injection was performed 1 hour after exposure once the above doses. The slaughter of animals was carried out at 7 days after irradiation (table 1).

Methods of statistical processing of the obtained results

For each row in the index is calculated arithmetic mean, standard error of the mean. The reliability of differences between subgroups was assessed using t - student criterion. Differences were considered significant at p<0,05.

The number of endogenous hematopoietic colonies of the claimed method and the prototype are shown in Table 2.

As can be seen from the analysis of this table the number of endogenous hematopoietic colonies in the spleen in comparison with the prototype increased by 47,3%.

Method of recovery of the spleen after radiation exposure, characterized in that laboratory mice one hour after irradiation is carried out intravenous allogeneic transplantation of multipotent mesenchymal stromal cells (MSCS) and hematopoietic stem cells (HSCs) obtained from the placenta of female mice during gestation 14 days, MSCS administered in a dose of 6.5 million cells/kg, and GCS in a dose of 400 thousand cells/kg.



 

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3 tbl

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