Vіsnik Naukovih Doslіdžen' (Apr 2018)
INDICATORS OF CELL CYCLE AND DNA FRAGMENTATION OF SPLEEN CELLS IN EARLY TERMS AFTER THERMAL BURNS OF SKIN ON THE BACKGROUND OF USING "LACTOPROTEIN WITH SORBITOL" OR HAES-LX-5 %
Abstract
In the scientific literature, there is no research data relative performance characteristics of the cell cycle and DNA fragmentation spleen cells after thermal skin burn on the background of the introduction of hyperosmolar infusion solutions. The aim of the study – is to establish the characteristics of the cell cycle and fragmentation of the cells of the spleen after 1, 3 and 7 days after burn injuries of the skin with the introduction of solutions of "lactoprotein with sorbitol" or HAES -LX -5 %. Materials and Metohods. The research was carried out on laboratory white rats males weighing 155–160 g, obtained from the vivarium of the Institute of Pharmacology and Toxicology of the Academy of Medical Sciences of Ukraine. The rats were divided into 6 groups in the experiment: 1, 2 and 3 groups – rats without thermal trauma, which were infused with 0.9 % NaCl solution, "lactoprotein with sorbitol" and HAES -LX -5% in dose 10 ml per kg. In the 4, 5 and 6 groups 0.9 % NaCl solution, "lactoprotein with sorbitol" and HAES -LX -5 % in a dose of 10 ml per kg after skin burning were administered to the rats. Burn skin damage was caused by applying to the pre-depilated lateral surfaces of the trunk of the rats for 10 seconds four copper plates (two plates on each side, each with a surface area of 13.86 cm2) which were preheated for 6 minutes in constant temperature water 100 ºC. Shaving of the lateral surfaces of the trunk of the rat, catheterization of the veins, staining of skin burns and decapitation of the animals was carried out under conditions of intravenous propofol anesthesia (at the rate of 60 mg/kg of animal weight). The DNA content of the rat spleen cell nuclei was determined by flow cytometry at the Partec multifunctional flow cytometer "Partec PAS ". UV radiation was used to stimulate DAP I fluorescence. From each sample of the nucleic suspension of the analysis subject to 20 thousand events. Cellular analysis of the cells was carried out using FloMax software (Partec, Germany) in full numeric matching according to the mathematical model, which determined: G0G1 – percentage ratio of G0G1 phase cells to all cells of the cell cycle (DNA content = 2c); S – percentage ratio of the phase of DNA synthesis to all cells of the cell cycle (DNA content > 2c and < 4c); G2 + M - percentage ratio of the G2 + M phase to all cells of the cell cycle (DNA = 4c); IP - the index of proliferation, which is determined by the sum of the indices S + G2 + M; BP – block of proliferation, which is evaluated by the ratio S / (G2 + M); SUB -G0G1 is an interval on the DNA histograms RN 1 before the peak G0G1, which indicates the cell nuclei containing DNA < 2c (DNA fragmentation detection). The statistical processing of the obtained results was carried out in the licensed package "Statistica 6.1" with the use of nonparametric methods for evaluating the results. Results and Discussion. In the application of the solution of "lactoprotein with sorbitol", after 1 day after burn skin damage, bigger mean values of the S-phase (39.4 %, p <0.05) were observed in comparison with the burn group after correction by 0.9 % NaCl solution, however, they remain significantly lower (by 35.4 %, p <0.05) relative to the mean values of this indicator in groups without burn injury. Also, higher values of the index of proliferation (38.6 %, p = 0.076) in the burn group + "lactoprotein with sorbitol" compared with the burn group + 0.9 % NaCl solution were observed. At the same time, when using HAES -LX -5 % against the background of skin burns, compared with similar indicators of the burn group + 0.9 % NaCl solution, the indexes of the G0G1 phases were significantly lower (by 4.8 %, p <0.05 ) and the interval of SUB -G0G1 (by 34.9 %, p <0.01) and higher S-phases (by 41.1 %, p <0.05) and the proliferation index (by 32.7 %, p < 0.05). When comparing the cell cycle parameters of the spleen cells between the groups of burn + "lactoprotein with sorbitol" and burn + HAES -LX -5%, after 1 day of the experiment, only a significant (p <0.05) by 30.3 % lower values of the SUB - G0G1 established when applied with HAES -LX -5%. Thus, after 1 day after burning the skin, "lactoprotein with sorbitol" had a more significant effect on synthetic processes, and HAES -LX -5 % – on processes both DNA synthesis and apoptosis compared with the use of 0.9 % NaCl solution. A similar picture of the nature of the effect of these drugs on the cell cycle parameters of the spleen cells was observed after 3 and 7 days after the burn injury of the skin. In particular, in comparison with the indicators of 0.9 % NaCl solution without burn after 3 days, on the background of burn and correction with the drug "lactoprotein with sorbitol" the average values of the proliferation block were higher, and on the background of correction with HAES -LX -5 % higher become the average values of the parameters of phase S and the block of proliferation and smaller – interval SUB -G0G1. It should be noted that only on the background of HAES -LX -5 % average values of the phase S indicators were significantly higher at the given time relative to the same indicator of burn groups + 0.9 % NaCl solution and burn + "lactoprotein with sorbitol". This indicates that at the time of the experiment HAES -LX -5 % more clearly contributed to the renewal of the cell population by stimulating DNA synthesis against the background of enhanced apoptosis in the background of burn injury. After 7 days after skin burn, there were no differences in the effect on the parameters of the cell cycle of the spleen cells between the drugs "lactoprotein with sorbitol" and HAES -LX -5 %. However, in both of these groups, higher mean values of the S-phase, the proliferation block and SUB -G0G1 intervals were found to be higher than the average values of similar burn group parameters + 0.9 % NaCl solution. Conclusions. The use of "lactoprotein with sorbitol" or HAES -LX -5 % solutions on the background of burn infections of the skin contributes to a more effective process for the renewal of the spleen cells by stimulating the synthesis of DNA and lower apoptosis level, especially with the use of HAES -LX -5 %.
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