The way to increase the functional activity of lymphocytes

 

(57) Abstract:

The invention relates to medicine, in particular to immunology. The method is carried out by exposure to helium plasma, obtained at an amperage of 30 And a voltage of 20 V and the gas flow of 2 l/min, the cells of the spleen, placed in medium RPMI-1640. The medium additionally containing 5% inactivated human serum 4 groups, 2 mm 1-glutamine, 10 mm buffer s, 510-5M 2-mercaptoethanol and 50 μg/ml gentamicin. Exposure is carried out for 20 s from a distance of 20 cm from the nozzle of the plasma torch. The method allows locally to affect the functional activity of lymphocytes in cell culture, and within the entire organism, the effect achieved in a short time. The method can be used in clinical practice as a method of stimulating effect on biological processes in cells and tissues. table 1.

The invention relates to medicine, in particular to immunology, and can be used in clinical practice as a method of stimulating effect on biological processes in cells and tissues.

The ability of lymphocytes to participate in immune reactions in organisms is Anna proliferation of lymphocytes.

There is a method of stimulating the functional activity of lymphocytes by an alternating magnetic field with a frequency of 100 Hz and magnetic field induction h-3T 1 and T, acting on the spleen cells in vitro for 30 and 60 minutes (N. E. Melnikova, A. S. Soloviev - Abstracts. Proceedings of the International conference "Ecology and life - 2000". Veliky Novgorod, 2000, S. 57).

In recent years in clinical practice has been widely used plasma flows. As a plasma-forming gas used is helium or argon. The flow of low-temperature plasma is used in the treatment of trophic ulcers, erysipelas, superficial suppurative and burn wounds. The positive effect of the plasma is associated with increased regenerative processes in wounds. An important role in regenerative processes play cells of the immune system (A. Babayev, Haematopoietic and lymphoid organs. //Structural bases of adaptation and compensation of disturbed functions /edited by D. S. Sarkisov. - M.: Medicine, 1987. - S. 328-342).

The method of increasing the functional activity of cells is that the cells of the spleen is placed in a medium RPMI-1640, optionally containing 5% inactivated human serum 4 groupview helium plasma, obtained at an amperage of 30 And a voltage of 20 V and the gas flow of 2 l/min. Exposure is carried out for 20 seconds from a distance of 20 cm from the nozzle of the plasma torch.

These optimal parameters were established in preliminary experiments. Other parameters of the gas flow, current and voltage, as well as the distance from the nozzle of the plasma torch to the object and the exposure time or did not modify the functional activity of lymphocytes, or had a suppressive effect. Additives to the medium RPMI-1640 ensured the viability of the lymphocytes. The ratio of the components was optimal.

Helium plasma provides a more rapid build-up of mitogen-induced lymphocyte proliferation compared with the action of the argon plasma Under the action of the helium plasma is increased and spontaneous proliferation of lymphocytes, whereas argon plasma, acting on cells of the spleen, leading to the suppression of spontaneous proliferation of lymphocytes.

The method is as follows.

As a source of lymphocytes using spleen cells of mice-hybrids (CBA x C57B1/6)F1 obtained from the nursery of the AMS "Pole". Fence lymphoid organs are produced in compliance with the rules of asepsis and anti solution of bleach, and then 96oethyl alcohol. Sterile extract spleen, cleaned of fat and connective tissue, placed in a chilled medium 199 and carefully crushed in a glass homogenizer. A suspension of lymphocytes filtered through a fine-meshed nylon, centrifuged for 7 min at 1000 rpm and decant the supernatant, the precipitate resuspended in the necessary volume of RPMI-1640 medium, supplemented with 5% inactivated human serum, 4 groups, 2 mm 1-glutamine, 2 mm Hepes buffer, 5 x 10-5M 2-mercaptoethanol, 50 μg/ml gentamicin. The viability of lymphocytes determine the camera Goryaeva in a mixture of 0.1% solutions Trypanosoma blue and eosin.

Under a light microscope MBI-3 count the number of living cells. Dead cells should be no more than 8-10%.

In experiments using plasma installation SUPR-M and physiotherapy plasma torch. 20x106studied spleen cells contribute in plastic vials in a volume of 1 ml of RPMI-1640 medium, optionally containing 5% inactivated human serum 4 groups, 2 mm 1-glutamine, 10 mm Hepes buffer, 5 x 10-5M 2-mercaptoethanol, 50 μg/ml gentamicin. Irradiation of lymphocytes hold helium plasma with an electric current of 30 a, a voltage of 20 V from a distance the human plasma stream within 20 seconds. All manipulations are performed on ice to eliminate thermal effect.

The functional activity of lymphocytes was assessed by the level of their spontaneous and PHA-induced proliferation in the reaction of besttransport.

The reaction besttransport perform micromodification (Horobryh centuries and others , 1983). Breeding mitogen PHA (Difko) cooked in medium RPMI-1640. Best practices, the concentration of mitogen determined in preliminary experiments. She was 31 ág/ml of medium cultivation. 5x105studied spleen cells contribute to the wells of microplates (Linbro) in a volume of 100 ál and add to it 100 ál of mitogen or RPMI-1640 medium. Microplasmin incubated in a thermostat at 37oC for 72 hours in an atmosphere containing 5% CO2.

For 16 hours before the end of the incubation the wells contribute 1 MK Ku3H-thymidine in a volume of 50 μl of RPMI-1640 medium. Then, using a 12-channel device, the sample is transferred onto the filters (Synpor-3, Czech Republic), sequentially washed wells saline solution, 5% trichloroacetic acid and 96othe alcohol.

The filters are dried, placed in scintillation fluid LGL-8 and register the number of pulses per minute on a beta-spectrometer.

For Statisticheskii t-criterion of student.

Example 1.

In the experience were taken 3 male hybrid (CBA x C57B1/6)F1. Mouse slaughtered by cervical dislocation, sterile extracted spleen. A suspension of lymphocytes was filtered through a fine-meshed nylon, centrifuged at 1000 rpm for 7 minutes. Then decanted supernatant and precipitate resuspendable in 2 ml of RPMI-1640 medium, supplemented with 5% inactivated human serum, 4 groups, 2 mm 1-glutamine, 10 mm Hepes buffer, 5 x 10-5M 2-mercaptoethanol, 50 μg/ml gentamicin. The viability of lymphocytes was determined in the Goryayev camera in a mixture of 0.1% solutions Trypanosoma blue and eosin 20x106the spleen cells were made in plastic vials in a volume of 1 ml of RPMI-1640 medium, supplemented with 5% inactivated human serum, 4 groups, 2 mm 1-glutamine, 10 mm Hepes buffer, 5 x 10-5M 2-mercaptoethanol, 50 μg/ml gentamicin. Lymphocytes were subjected to helium plasma, obtained at an amperage of 30 And a voltage of 20 V and the gas flow of 2 l/min. effect on the lymphocytes was carried out in 20 seconds from a distance of 20 cm from the nozzle of the plasma torch. The controls were cells of the spleen, have not been exposed to helium plasma All manipulations were performed on ice to eliminate thermal effect 5x105and the And or RPMI-1640 medium. Check cell response was implemented to enable3H-thymidine into DNA proliferous cells.

Experiments have shown that irradiation of the spleen cells helium plasma for 20 seconds resulted in increased spontaneous and PHA-induced lymphocyte proliferation (table).

The test method was performed on 12 male hybrids (Swahs/6)F1. In each series of experiments, spleen cells were pulirula from 3-4 animals 20x106the spleen cells were made in plastic vials in a volume of 1 ml of RPMI-1640 medium, supplemented with 5% inactivated human serum, 4 groups, 2 mm 1-glutamine, 10 mm buffer Hpes, 5x10-5M 2-mercaptoethanol, 50 μg/ml gentamicin.

Has identified the following groups:

1. The control group cells of the spleen, have not been exposed to helium plasma.

2. Experimental group - spleen cells exposed to helium plasma for 20 seconds.

After irradiation studied the functional activity of lymphocytes in reaction besttransport. Studies have shown that exposure to helium plasma cells enhances their spontaneous and PHA-induced proliferation.

The results of the experiments the t lymphocytes provides the following benefits:

1. Compared with other ways of stimulating the functional activity of lymphocytes, the effect is achieved as a result of very short duration factor (20 sec).

2. The method allows locally to affect the functional activity of lymphocytes in cell culture, and within the entire organism.

The way to increase the functional activity of lymphocytes in medium RPMI-1640, optionally containing 5% inactivated human serum 4 groups, 2 mm 1-glutamine, 10 mm buffer s, 510-5M 2-mercaptoethanol, 50 μg/ml gentamicin, by physical impact factor, characterized in that as a physical factor using a helium plasma, obtained at an amperage of 30 And a voltage of 20 V and the gas flow of 2 l/min, and exercise effects on lymphocytes within 20 s from a distance of 20 cm from the nozzle of the plasma torch.

 

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FIELD: biology, genetic engineering.

SUBSTANCE: invention relates to preparing immortalized cellular lines from health human skin tissues and can be used in immunological, pharmacological, photo- and chemical-toxicological analysis of cutaneous response, for expression of heterologous genes and for construction of artificial skin. Keratinocytes are immortalized by infection of keratinocytes of health human. The human skin sample is isolated and prepared its for culturing in vitro. Keratinocytes are prepared from this prepared human skin sample and plated in serum-free medium for growing keratinocytes in cultural plates with cover alleviating attachment and growth of cells. In the process for culturing keratinocytes the serum-free medium is replaced to provide preparing the optimal confluent growth of cells in culture with continuous maintenance of cup cover. Keratinocytes are transferred in selective serum-free medium in cultural cups with cover and infected with vectors pLXSHD + SV40(#328) and pLXSHD + E6/E7. Then prepared immortalized keratinocytes are transferred in cultural cups with cover to useful medium for proliferation. Then prepared proliferated keratinocytes are transferred in medium with high calcium content for differentiation in cultural chambers with cover. Invention provides preparing the human keratinocyte cellular line that has no oncogenic property and retains capacity for differentiation and expression of proteins and enzymes expressing by normal differentiated keratinocytes being even after increased number of passages in culture. Also, this cellular line forms lamellar and polarized epithelium with keratinized layer (stratum corneum) consisting of ortho-keratinocytes in the process for culturing in organotypical culture in serum-free medium and without layer of feeding cells.

EFFECT: improved immortalizing method, valuable biological properties of cellular line.

7 cl, 2 dwg, 4 ex

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