CD4^+ CD25^- T细胞凋亡机制的研究

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

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。

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 ,CCD4^＋CD25^- T cells from BALB/e mice were stimulated by anti-CD3 and anti-CD28 antibody, and CD4^＋CD25^-T cells from DOI 1.10 mice were activated by OVA323-339 plus APC. The apoptosis gene array was developed to detect the apoptosis-related gene expression inCD4^＋CD25^- - T cells and CD4^＋CD25^- T cells. Flow eytometry 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 OVA323-339 peptide plus APC was significantly inhibited by either anti-FasL neutralizing antibody or anti-TRAIL neutralizing antibody, and zVAD-fmk as well. Conclusion：FasL, TRA/L and other apoptosis-related molecules might be involved in the AICD of CD4^＋CD25^-T cells.