Method for recovery and preparation of leukaemia cell suspension from spleens of inbred mice akr/jy

FIELD: medicine.

SUBSTANCE: invention refers to medicine and biology, more specifically to technology and methods for recovery of leukaemia cell suspension from spleens of high-leukaemia mice, and can be used in experimental oncological haematology for storage of low-frequent subtypes of murine leukaemia. There is offered a method for preparing a leukaemia cell suspension from spleens of inbred mice AKR/JY involving skin preparation with an antiseptic, splenectomy, decapsulation and homogenisation of spleen, filtration of the prepared suspension to be diluted in a sterile cooled artificial extracellular medium to the required concentration of leukaemia cells, prepackaging of the prepared suspension in ampoules for storage and transportation with decapsulation, grinding and homogenisation of fragmented spleen pulp performed in polyglucin solution containing ozone 25.72 mg/l and cooled to 24.01.0C, leukaemia cells are filtered and diluted in polyglucin solution containing ozone 25.72 mg/l and cooled to 15.02.0C.

EFFECT: invention provides maximum safety of specific functional adequacy of the transplanted leukaemia cells.

1 ex, 1 tbl

 

The invention relates to medicine, namely technology selection and preparation of suspensions of leukemic cells from the spleens of mice vysokoklasnyh lines, and can be used in experimental Hematology for the preservation of rare subtypes of leukemia mice.

The positive results of inoculation of leukemia is only possible under conditions of maximum security baseline specific functional usefulness of the transplanted leukemic cells. The presence of microorganisms and their toxins, and tissue aggregates in transplantable material damage transplantable leukemia cells, reduces the quality of the prepared suspension and leads to negative results inoculation of a certain type (subtype) leukemia mouse.

There is a method of selection and the preparation of suspensions of leukemic cells from spleens line of mice in which all manipulations are performed under aseptic conditions, which before the opening of the abdominal wall skin and hair in the projection laparotomic incision is treated with antiseptic (alcohol, iodine) [Workshop on immunology. The textbook. book for students. the Supreme. the textbook. institutions / Iiandreview [and others]- 2nd ed., Corr. and supplementary): Izdat. Center "Academy", 2004. - p.17-24]. This method does not provide a full waste is arovane infection on the body of the test animal, which falls into the suspension transplantable leukemia cells and inhibits (partially or fully) their specific functional completeness.

There is a method of allocation of leukemia cells in chilled artificial extracellular environment sterile saline [Rauschenbach NO Experimental studies of leukemia / Maurauding. - M.: Publishing House."Medgiz", 1956. - 184 S.]. The disadvantage of this environment is the ability to cause irreversible aggregation of leukemia cells and debris from the formation of stable clusters, and the absence in it of antiseptics and preservatives that can disinfect and protect transplantable cells from irreversible damage during an extended stay outside the body at positive temperature.

In modern medicine to improve the antimicrobial activity of the organism are increasingly using ozonated officinal fluid. It is connected with disinfectant, detoxifying, bacteriostatic, bactericidal, antiviral and antifungal effects of ozone ("methods of application of ozone in medicine" (methodical recommendations) IPELA and other Kyiv, 2004, S.-23). The ozonized solution poliglyukina, which has a disinfecting and disaggregated action and properties of the cell preservative, technology selection and preparation suspe the Ziya leukemia cells were not used (the Study of the bactericidal properties and sterility ozonated official liquids / A.I. Korabelnikov, Auetayev, Iaaciaeiinoaa // Annex to NMI Ozone therapy. - 2003. - P.41; Cryoconserved / Amelius, Migrage, Nuser. - K.: Science. Dumka, 1974. - 200 S.; Medicines (manual for doctors) / Medmaravis. - M.: Publishing House. "New wave", 14th edition, 2nd volume. - 2000).

For the prototype of the present invention is selected known method of selection and the preparation of suspensions of leukemic cells from the spleens of linear mice, including the treatment of skin antiseptic, splenectomy, decapsulation and homogenization of the spleen, filtering the resulting suspension and dilution in sterile chilled artificial extracellular environment to the required concentration leukemia cells, filling prepared suspension in vials for storage and transportation. After preparation of the suspension transplantable material should be used promptly or frozen (Workshop on immunology. The textbook. book for students. the Supreme. the textbook. institutions / Iiandreview [and others] - 2nd ed., Corr. and supplementary): Izdat. Center "Academy", 2004. - p.17-19). This method is the closest technical solution to offer. The disadvantage of this method is that the most isolated from spleens of leukemic cells outside the body to lose specific functional completeness is the ability to previati defined the ID (subtype) leukemia healthy mouse vysokochastotnoi line.

The task of the invention is the provision of maximum security specific functional usefulness of the transplanted leukemic cells.

The problem is solved in that the stages of selection and preparation of the suspension transplantable leukemia cells using chilled ozonated solution poliglyukina.

The present invention meets the criteria of "novelty" and "inventive step", because in the process of conducting patent information research has not identified sources of information prejudicial to the novelty of the proposed method.

Our proposed method consists in the gradual separation from spleens line of mice and preparation of suspension of leukemia cells in chilled ozonated solution poliglyukina.

Our studies show that the ozonized solution poliglyukina retains the biological properties of the most isolated from the spleens of mice leukemia cells and high positive perelivania a specific type (or subtype) of leukemia, while the application in the same terms saline without antiseptic support ozone and the absence of cell preservative does not retain the biological properties of most transplantable cells with subsequent high negative modeling determined the n type (subtype) of leukemia mice.

Experimental studies performed in the FGI "Kirov research Institute of Hematology and blood transfusion and gynecology".

The effectiveness of the method was tested in 16 mice of inbred AKR/JY. The results of the study are given in the table.

Conclusions

Selection and preparation of suspension leukemia cells offer a way into a chilled ozonated solution poliglyukina retains the biological properties of the majority of leukemic cells outside the body at the optimal temperature for a long time and provides a high percentage of perelivania a specific type (or subtype) of leukemia mouse inbred lines.

Example.

The method is carried out in aseptic conditions in a sterile mask, sterile gloves, sterile ophthalmic surgical instruments (scissors, forceps, scalpels), sterile prepravljenom table with needles, sterile tissue grinder, using a 4-layer nylon filter, sterile conical tube 10 ml, officinal preparation "Poliglyukin in 400 ml glass vial containing 25,72 mg/l ozone, needle-vodusek system for transfusion of blood, blood products and intravenous fluids single use, water thermometer, open at the top of the vessel from the mixer is fed running water with a temperature from 24,01.0C is about 15,02.0, the ozone generator system of polymer tubes and ozone destructor, vials with a volume of 5-10 ml for packaging prepared suspension of leukemia cells.

The preparatory stages.

Ozonation solution poliglyukina. The method is as follows: the ozone generator is connected to a source of oxygen and in the grid, set on the instrument panel the ozone concentration in the ozone-oxygen mixture (ACS) at the outlet of the ozonizer 10 to 30 mg/l In the tube set of three needles, through one of which served the ACS, the second gets ozonated poliglyukin (OP), and through a third Mature OAKS removed

Table
Comparative results of inoculation of a suspension of leukemia cells, selected and prepared with known and proposed methods
No.Type of leukemiaThe result of the inoculation suspension leukemia cells, selected and prepared in various ways
Prototype methodThe proposed method
1.myeloblastic leukemianegativemalob asty leukemia
2.T-cell lymphoblastic leukemianegativeT-cell lymphoblastic leukemia
3.T-cell lymphoblastic leukemianegativeT-cell lymphoblastic leukemia
4.T-cell lymphoblastic leukemiaT-cell lymphoblastic leukemiaT-cell lymphoblastic leukemia
5.prolymphocytic leukemianegativeprolymphocytic leukemia
6.T-cell lymphoblastic leukemianegativenegative
7.T-cell lymphoblastic leukemianegativeT-cell lymphoblastic leukemia
8.T-cell lymphoblastic leukemianegative T-cell lymphoblastic leukemia
9.myeloblastic leukemianegativemyeloblastic leukemia
10.T-cell lymphoblastic leukemianegativeT-cell lymphoblastic leukemia
11.T-cell lymphoblastic leukemiaT-cell lymphoblastic leukemianegative
12.T-cell lymphoblastic leukemianegativeT-cell lymphoblastic leukemia
13.B-cell lymphoblastic leukemianegativeB-cell lymphoblastic leukemia
14.T-cell lymphoblastic leukemianegativeT-cell lymphoblastic leukemia
15.B-cell lymphoblastic leukemia B-cell lymphoblastic leukemiaB-cell lymphoblastic leukemia
16.prolymphocytic leukemianegativeprolymphocytic leukemia

in the destructor. The end of the long needle is set on the bottom of the vial. Sparging poliglyukina ACS start 15 min before the experiment and then continue all the time infusion that provides consistency TO the OP. The effectiveness of the antimicrobial activity of OP depends on the concentration of ozone in solution. In the course of the research sample OP was created around made "Museum" crops like gram-positive and gram-negative microflora zone sterility in diameter from 8 to 10 mm, an average of 9.40.7 mm, that is, had a distinct bactericidal effect and sterility in 24 hours.

The cooling of the bottle with a solution poliglyukina. Temperature OP during the experiment decreases from 24,01.0C to 15.02,0C. For this bottle with polyglycine give the position of the tube up and place it in the glass of the mixer is supplied with running water set temperature.

After sleep (ether, chloroform) adult mice (weighing 18-22 g body temperature of 381C) with advanced leukemia (4 to 10 months from the start of the experiment), the carcass is placed on preaproval the second table on the right side, fixed to the table with a sterile needle, then alcohol is treated with the left side. In the "waist" of the animal scissors make a transverse skin incision length 2-2 .5 cm to the left from the middle of the trunk, then cut muscles, tweezers removed from the abdominal cavity spleen maroon color weight 700300 mg, cut off its connective fibers and placed in a sterile Petri dish, in which the polymer backbone drip fed OP, cooled to 24.01,0C. under these conditions, the spleen is freed from residues of connective tissue (decapsulated), after which the pulp of the spleen scissors fragmenting into small pieces and homogenized. Homogenized filtered through one or two nylon 4-ply filter into a test tube, in which the polymer backbone drip fed ozonated solution poliglyukina cooled to 15.02.0and dilute them to a concentration of from 1106up to 1107cells/ml. After that, the prepared suspension leukemia cells Packed in vials for storage and transportation. The safety of specific functional usefulness of the transplanted leukemic cells are determined by the results of the inoculation of a given IDA (subtype) of spontaneous leukemia.

Studies show that out of the 32 cases of inoculation of a certain type of leukemia positive results when using the way-is rototype received 3 or 18.75% of cases, while the application of the proposed method gave positive results in 14 or 87.5% of the cases. The difference being statistically significant (P<0,05).

The proposed technology selection and preparation of suspensions of leukemic cells from the spleens of linear mice provides stable high positive result of the inoculation of a given subtype of leukemia and can be used in institutions of medical and biological profile in experimental Hematology for the preservation of rare subtypes of leukemia mice.

Isolation and preparation of suspensions of leukemic cells from the spleens of mice of inbred AKR/JY, including the treatment of skin antiseptic, splenectomy, decapsulation and homogenization of the spleen, filtering the resulting suspension and dilution in sterile chilled artificial extracellular environment to the required concentration leukemia cells, filling prepared suspension in vials for storage and transport, wherein decapsulation, grinding and homogenization of the pulp of the spleen is performed in solution poliglyukina containing 25,72 mg/l ozone and cooled to (24,01,0C, filtration and dilution of the suspension leukemia cells perform in solution poliglyukina containing 25,72 mg/l ozone and cooled to (15,02,0)C.



 

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