CD4+ CD25+调节性T细胞AICD机制的研究

目的探讨CD4^＋CD25^＋调节性T细胞活化诱导的细胞死亡（AICD）发生的机制。方法CD4^＋CD25^＋T细胞以磁性细胞分离器（MACS）从BALB／c小鼠或DO11．10小鼠的静息T细胞分离纯化。体外细胞增殖抑制实验证实其免疫调节作用。CD4^＋CD25^＋T细胞的AICD以CD3／CD28单克隆抗体活化或以特异性OVA323-339肽、抗原提呈细胞活化等两种方法获得。CD4^＋CD25^＋T细胞凋亡相关基因的表达通过实时定量PCR检测。流式细胞仪检测细胞的凋亡率。进一步观察FasL中和抗体、TRAIL中和抗体及caspase抑制剂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％，该细胞可特异性表达Foxp3基因，能明显抑制效应性T细胞的体外增殖。CD3／CD28抗体以及OVA特异性抗原活化8d的CD4^＋CD25^＋调节性T细胞AICD达39％～45％。活化前后的CD4^＋CD25^＋调节性T细胞死亡受体家族表达发生明显变化；FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4^＋CD25^＋调节性T细胞的凋亡。结论FasL／Fas及其他凋亡相关分子可能参与了CD4^＋CD25^＋调节性T细胞的凋亡。

Activation induces cells death(AICD) of CD4+ CD25+ regulatory T cells

Objective To investigate the mechanism of AICD in CD4+ CD25+ regulatory T cells(Treg). Methods Murine CD4+ CD25+ T cells From naive BALB/c or DO11.10 mice were isolated by MACS CD4+ CD25+ Treg cells from BALB/c mice were stimulated with anti-CD3/CD28 antibodies, and those from DO11.10 mice were incubated with OVA323-339 peptide plus APC respectively. Real time PCR was applied to detect the gene expression pattern in CD4+ CD25+ Treg cells. Flow cytometry was performed to determine the apoptosis of CD4+ CD25+ Treg cells. The influence of anti-FasL, anti-TRAIL neutralizing antibody and zVAD-fink on AICD of CD4+ CD25+ Treg, cells was evaluated. Results CD4+ CD25+ T cells were classified by MACS with 98% purity and specifically express the Foxp3 gene which is the 'master control gene' for Treg cells and could suppress the proliferation of CD4+ CD25+- T cells. The apoptosis of freshly isolated CD4+ CD25+ Treg cells was less than 5% , but could reach to 39%-45% after being activated in vitro for 8 days. The results of real time PCR showed different gene expression pattern of TNF family member in freshly isolated CD4+ CD25+ Treg cells and activated CD4+ CD25+ Treg cells. The apoptosis of CD4+ CD25+ Treg cells which induced by anti-CD3/CD28 antibody or OVA323-339, peptide plus APC was significantly inhibited by either anti-FasL neutralizing antibody, or anti-TRAIL neutralizing antibody and caspase inhibitor zVAD-fmk as well. Conclosion FasL/Fas and other apoptosis-related molecules might be involved in the AICD of CD4+ CD25+ Treg cells.