Interleukin-35 Promote The T Lymphocyte Apoptosis Mechanisms’ Research

Objective:1. To investigate the effect of T lymphocyte apoptosis after the IL-35 co-cultured with Jurkat.2. Discussion the time/dose relationship for IL-35-mediated apoptosis of T lymphocytes.3. Discussion the mechanism of IL-35 to promote the T lymphocyte apoptosis.Methods:Using the 10%FBS RPMI1640 to culture the Jurkat cell. Replacing the culture medium per 48h and cell passaging per 72h. Preparing the 1×106 cells of magnitude for experiment.Collecting the cells of 1×106/ml to seed in 12-well cell culture plate. Using the different concentration (10ng/ml,80ng/ml,640ng/ml) of Interleukin-35(IL-35) to stimulate the Jurkat T lymphocytes by 24h and 36h. Then collecting the cells to divide into N group (Normal), C group(Control),24h group, lOng/ml group,80ng/ml group.1.Using the FITC apoptosis detection kit produced by BD company of American for testing the apoptosis for Jurkat cell by Flow cytometry. Interoperating the effect of apoptosis by Statistical analysis.2. Choosing the 24h lOng/ml group,80ng/ml group,640ng/ml group to test the protein situation of Bcl-2 and the AktSer473 phosphorylation sites situation. Statistical analysis the results and investigate its’ significance. Using the caspase-3,8,9 ELISA assay kit produced by the company of eBioscience, test the protein changing situation of caspase-3,,8,9 that above groups samples.3. Using the Fluo-3 AM calcium probe to detect the concentration of Ca2+ from the above groups samples, and statistical analysis them.Results:1.IL-35 promote T lymphocyte apoptosis, and there is a time/dose relationship. The results show that IL-35 co-cultured with Jurkat T cells for 24h,36h compared to the normal group (N) and control group (C) the apoptosis rate increased (P <0.01).24h,36h group Using lOng/ml concentration of IL-35 stimulation, compared with the N group and C group, there is a phenomenon of apoptosis rate increased (P <0.01). The lOng/ml group and 80ng/ml group of apoptosis in 36h was higher than 24h group (P<0.01). The 640ng/ml group apoptosis rate in 24h group was higher than 36h group (P<0.01). At 24h, with increasing concentrations of IL-35 (80ng/ml,640ng /ml) the apoptosis rate showed that upward trend compared with the N group and C group(P<0.01).The 640ng/ml group apoptosis rate get highest, Figure 1. At 36h, the apoptosis rate get highest in 80ng/ml, Figure 1.2.Detection 24h, 10ng/ml group, 80ng/ml group,640ng/ml group,Bcl-2 and AktSer473 phosphorylation.10ng/ml group Bcl-2 protein was decreased than Control group (P<0.01).640ng/ml group, Bcl-2 protein expression compared with Control group was significantly decreased and reached the lowest value(P<0.01).80ng/ml group,640ng/ml group,AktSer473 phosphorylation compared with Control group was significantly inhibited, and 640ng/ml group AktSer473 phosphorylation expression get lowest(P<0.01).3. Detection 24h,10ng/ml group,80ng/ml group,640ng/ml group protein expression of Caspase-3,8,9.Comapred with the N group and C group from 10ng/ml begin to upward,640ng/ml group get the highest value(P<0.01).Caspase-8 is hardly expressed in this process of apoptosis, Figure 7.4. Testing the intracellular of Ca2+ concentration. We found that the intracellular Ca2+ concentration of 640ng/ml group get highest compared with Control group.Conclusion:1.Different concentrations of IL-35 can promote apoptosis of Jurkat cell in different time.2. The intracellular of Ca2+ concentration, Caspase-3,8,9, Bcl-2,and AktSer473 phosphorylation were exist relationship in different concentration of IL-35 stimulate the T lymphocyte apoptosis apoptotic process.3. Mitochondrial apoptotic pathway may be have a important effect on IL-35 promote T lymphocyte apoptosis pathway.