CD4+CD25-T细胞凋亡机制的研究

目的：探讨CD4^＋CD25^-T细胞AICD发生的机制.方法：磁性细胞分离器（MACS）分离CD4^＋CD25^- T细胞.以CD3/CD28单克隆抗体活化BALB/c小鼠CD4^＋CD25^-T细胞或以OVA323-339肽、抗原递呈细胞活化DO11.10小鼠CD4^＋CD25^-T细胞两种方式建立AICD模型.基因芯片检测CD4^＋CD25^-T细胞和CD4＋CD25＋T细胞凋亡相关基因的表达.流式细胞仪检测细胞的凋亡率.并观察FasL中和抗体、TRAIL中和抗体及zVAD-fmk对CD4^＋CD25^-T细胞凋亡的影响.结果：MACS成功分离CD4^＋CD25^-T细胞,纯度可达98%.建立了CD3/CD28抗体以及OVA特异性抗原活化的CD4^＋CD25^-T细胞AICD模型,CD4^＋CD25^-T细胞凋亡率达35%～40%.基因芯片分析发现CD4^＋CD25^-T细胞相对高表达TRAIL、FAS,而CD4^＋CD25^-T细胞相对高表达DR5、FasL.FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4＋CD25＋T细胞的凋亡.结论：FasL、TRAIL及其它凋亡相关分子可能参与了CD4^＋CD25^-T细胞的凋亡.

Study on the mechanism of activation induced cells death(AICD) in CD4+CD25-T cells

Objective:To investigate the mechanism of AICD in CD4~+CD25~-T cells.Methods:Murine CD4~+CD25~-T cells were isolated by MACS(magnetic cell sorting).To induce AICD,CD4~+CD25~-T cells from BALB/c mice were stimulated by anti-CD3 and anti-CD28 antibody,and CD4~+CD25~-T cells from DO11.10 mice were activated by OVA_(323-339) plus APC.The apoptosis gene array was developed to detect the apoptosis-related gene expression in CD4~+CD25~-T cells and CD4~+CD25~+T cells.Flow cytometry was performed to determine the apoptosis of CD4~+CD25~-T cells.The influence of anti-FasL,anti-TRAIL neutralizing antibody,and zVAD-fmk on AICD of CD4~+CD25~-T cells was evaluated.Results:CD4~+CD25~-T cells could be successfully sorted by MACS with 98% purity.The apoptosis of freshly isolated CD4~+CD25~-T cells was <5%,but the apoptosis of CD4~+CD25~-T cells activated in vitro for 8 days reached to 35%-40%.The results of apoptosis gene array showed different gene expression pattern in CD4~+CD25~-T cells and CD4~+CD25~+T cells.The apoptosis of CD4~+CD25~-T cells which was induced by anti-CD3/CD28 or OVA_(323-339) peptide plus APC was significantly inhibited by either anti-FasL neutralizing antibody or anti-TRAIL neutralizing antibody,and zVAD-fmk as well.Conclusion:FasL,TRAIL and other apoptosis-related molecules might be involved in the AICD of CD4~+CD25~-T cells.