CD3TCR复合物抗体诱导不成熟胸腺细胞亚群的凋亡

Apoptosis effect of induced by antibodies to CD3/TCR complex in immature T cells

目的 :采用AnnexinV ,PI染色 ,流式细胞仪分析的方法、DNA琼脂糖电泳法检测不同CD3 TCR复合物抗体对小鼠胸腺T淋巴细胞亚群的促凋亡作用。方法 :新鲜分离胸腺细胞 ,加入PMA、anti TCR +anti CD2 8mAb ,anti TCRmAb等培养 2 0小时 ,AnnexinV ,PI ,CD4 ,CD8细胞染色以及TCR三染 ,进行FACS分析 ,同时抽提培养细胞DNA进行琼脂糖电泳。结果 :PMA +IONO诱导胸腺T细胞的凋亡作用最强 ,anti TCR +anti CD2 8mAb次之 ,anti TCRmAb最弱。结论 :AnnexinV ,PI细胞染色 ,流式细胞仪分析的方法可以灵敏检测T淋巴细胞的凋亡 ;anti TCR +anti CD2 8mAb能明显增强anti TCRmAb促不成熟的CD4 + CD8+TCR+ 和CD4 + TCR+ 细胞的凋亡作用 ;不成熟T细胞经TCR与自身抗原结合的活性过程能产生克隆删除从而产生自我耐受。

Objective:To investigate the apoptosis induced by antibodies to CD3/TCR complex in immature T cells, lymphocytes were labelled with AnnexinV,PI,then analyzed by flow cytometry and DNA were extracted agarose electrophoresis Methods:Fresh thymocytes and lymph node cells were separated, then the cells were cultured with the addition of PMA?anti TCRαβ+ anti CD28 mAb and anti TCRαβ up to 20 h, stained with AnnexinV,PI; CD8 FITC,CD4 PE,TCR APC,the data were collected through FACS analysis ; DNA was isolated from cultured thymocytes, and analyzed through agarose electrophoresis Results:Effects of different stimulators on the apoptosis of thymocytes are different PMA plus IONO is the most intensive stimulators in thymocyte apoptosis, anti TCRαβ is the weakest Conclusion:The method that FACS analysis in combines with AnnexinV,PI staining permits the detection of apoptosis cells at sensitive level; anti TCRαβ+ anti CD28 mAb induces significant apoptosis of immature CD4 +CD8 +TCR + and semimature CD4 +TCR + thymocytes than anti TCRαβ mAb alone;Activation of this process in immature T cells by the binding of the TCR to self antigens may therefore be the mechanism which produces clonal deletion and consequently self tolerance