线粒体途径在肿瘤坏死因子凋亡诱导配体诱导结肠癌细胞凋亡中的调节作用

Study on the effects of mitochontrial pathways on apoptosis in colon carcinoma cells induced by tumor necrosis factor related apoptosis inducing ligand

目的探讨线粒体途径在肿瘤坏死因子凋亡诱导配体(TRAIL)诱导结肠癌细胞凋亡过程中的调节作用,为临床合理用药提供理论指导。方法采用流式细胞仪技术、荧光显色技术和Western印迹技术检测TRAIL处理结肠癌细胞SW1116后,在不同时间细胞凋亡情况、线粒体完整性改变(ΔΨm、cardiolipin情况)以及线粒体下游通路细胞色素C和Caspase-9的表达情况。结果TRAIL诱发结肠癌细胞凋亡,于4 h达凋亡高峰,凋亡指数为32．98%；在4 h出现线粒体ΔΨm下降和cardiolipin丢失增加,造成其内膜损伤；细胞色素C表达及Caspase-9酶活性随时间的延长而增加,24 h酶活性达到最大峰值为(48．12±2．21)μmol·L^(-1)·h^(-1)·mg^(-1)蛋白。TRAIL诱导的线粒体损伤可被Caspase抑制剂Z-VAD．fmk所抑制。结论线粒体途径参与TRAIL诱导结肠癌细胞的凋亡过程,以Caspase依赖方式引发线粒体ΔΨm和cardiolipin丢失,造成内膜损伤,导致细胞色素C释放和Caspase-9激活,诱发凋亡。

Objective To explore the effects of mitochondrial pathways on apoptosis in colon carcinoma ceils induced by Tumor necrosis factor related apoptosis inducing ligand and offer evidences for TRAIL application in clinic. Methods Apoptosis, integration of mitochondria （including △ψm, cardiolipin）, activity of Caspase-9 and release of cytochrome c in colon carcinoma ceils SW1116 treated with TRAIL, were detected by means of flowcytometry, flurometer method and western-blot at the different time point. Results After treated with TRAIL for 4 hours, the apoptosis index was 32.98%, and the damage of mitochondria occurred with △ψm, cardiolipin decreased, and the activity of Caspase-9 and cytochrome c increased. The Caspase-9 activity at 24 hour was （48.12±2.21） μmol·L^-1·h^-1·mg^-1 protein. Mitochondrial damage induced by TRAIL could be inhibited by Caspase inhibitor Z-VAD. fmk. Conclusion Mitochondrial pathways involved in the apoptosis of colon carcinoma ceil induced by TRAIL. Cytochrome c was released and Caspase-9 was activated in the Caspase-dependent manner after the damage of mitochondfial.