Experiment Of The Effect Of KRP203 On CD4~+T Lymphocytes Immune Function In Rats

Objective: To investigate the effect of KRP203 on the proliferation, differentiation and migration function of CD4+ T lymphocytes in rats.Methods: Proliferation: Magnetic bead sorting of SD rat spleen cells to obtain CD4+T lymphocyte, Coated CD4+T lymphocytes by CD3 monoclonal antibody stimulation for 48 hours,and so they are in a state of activation. According to the KRP203 concentration in the medium of different, the experiments were divided into four groups: Control group, 10ng/ml group, 100ng/ml group, 1000ng/ml group. 5 days later, then use CCK8 detection of CD4+T lymphocyte proliferation. Differentiation:Culture 2 days group :Stimulation activation method of cells was the same as above. Add different concentration gradient of KRP 203 to activated cells Flow cytometry analysis of different experimental group CD4+CD25+Treg cells ratio after culture for 2 days.So as the 5 days groups.QPCR assay each group m RNA expression of S1P1 and Fox P3 in CD4+T cells. QPCR assay each group Th1 cell transcription factor T-bet m RNA and Th2 cell transcription factor Gata-3 m RNA expression. QPCR assay each group m RNA expression of the Th1, Th2 cells related cytokines, such as IL-2, TNF- alpha, IL-4, IL-5 and IL-10. Detect the cell supernatant of IL-2, IL-4, IFN- gamma cytokine concentrations with ELISA kit. Migration: The activated CD4+T lymphocyte was placed in the upper Transwell chambers, and culture with different concentrations and 100ng/ml SDF-1 in the 24–well plate. Put them in the 5%CO2, 37 DEG C incubator culture for 4h. Counting of different experimental groups of CD4+T lymphocyte migration.Results: In the proliferation results we found that compared with the control group no significant difference on cell proliferation rate changes,though CD4+T cells were cultured with various dose concentration of KRP203.In the differentiation results we found that no significant difference on CD4+CD25+Treg cells ratio in the 2 days group. 5 days later, the 1000ng/ml groups increase the ratio of CD4+CD25+Treg cells. The expression of drug treatment group of S1P1 m RNA and Fox P3 m RNA has no statistically significant difference between the groups. The expression of 1000ng/ml treatment group of T-bet m RNA reduced compared with the control group, while Gata-3 m RNA increased in 100ng/ml group and 1000ng/ml group than thecontrol group. The 1000ng/ml treatment group of Th1 cell related cytokines IL-2m RNA expression compared with the control group was reduced. Th2 related cytokine IL-4, IL-5m RNA expression increased in 100ng/ml group and 1000ng/ml group than the blank group. There was no difference in each group of IL-10 m RNA expression. The results of Elisa detection shows that 1000ng/ml treatment group of Th1 related cytokines IL-2 and IFN-γ concentration lower than the blank group, while Th2 related cytokines IL-4 are elevated in100ng/ml group and 1000ng/ml group than blank group. We find that 100ng/ml group and 1000ng/ml group can induce the migration of the CD4+T cell. There were statistically significant differences in cell count compared the control group.Conclusions: KRP203 inhibit the role of immune tolerance by induce the differentiation of CD4+T cells to Th2 cells and inhibit T cell migration.