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本研究探讨了外源性C2-神经酰胺诱导人结肠癌HT-29细胞凋亡中,线粒体膜间隙凋亡蛋白的释放机制.不同浓度C2-神经酰胺作用HT-29细胞,流式细胞仪检测线粒体膜电位(△Ψm),线粒体/细胞液分离试剂盒分离亚细胞成分,聚丙烯酰胺凝胶电泳检测细胞色素C(Cytc)、高温必需蛋白A2(HtrA2)、线粒体源性半胱天冬氨酸蛋白酶第二活化因子(Smac)、凋亡抑制蛋白(XIAP)和半胱天冬氨酸蛋白酶-3(Caspase-3)蛋白表达水平.实验结果显示25和50μmol/LC2-神经酰胺作用细胞6h,△Ψm即开始下降(P<0.05),且环孢霉素能通过调节线粒体膜通透性转换孔抑制△Ψm的下降.C2-神经酰胺对Cytc,HtrA2和Smac总蛋白表达没有明显影响,但能诱导Cytc,HtrA2和Smac从线粒体释放入细胞液中,并下调XIAP蛋白的表达及活化Caspase-3.在Caspase抑制剂存在下,C2-神经酰胺仍能诱导Cytc和HtrA2从线粒体释放,但不能诱导Smac释放.因此认为C2-神经酰胺能通过线粒体凋亡通路诱导HT-29细胞凋亡,C2-神经酰胺诱导Cytc和HtrA2从线粒体的释放是Caspase非依赖性的,而Smac释放是Caspase依赖性的.
This study investigated the release mechanism of exogenous C2-ceramide in the apoptosis of human colon cancer HT-29 cells, and the mitochondrial gap-gap protein was detected by flow cytometry in C2-ceramide-treated HT-29 cells Mitochondrial membrane potential (△ Ψm), mitochondria / cytosolic separation kit separation of subcellular components, polyacrylamide gel electrophoresis of cytochrome C (Cytc), high temperature essential protein A2 (HtrA2), mitochondrial-derived caspase The expression of Smac, XIAP and Caspase-3 in the cells of 25 and 50 μmol / L Ce2 + (P <0.05), and cyclosporine inhibited the decrease of △ Ψm by regulating the mitochondrial membrane permeability transition.C2-ceramide had no obvious effect on the expression of Cytc, HtrA2 and Smac, But can induce the release of Cytc, HtrA2 and Smac from the mitochondria into the cytosol and down-regulate the expression of XIAP protein and activate Caspase-3. In the presence of Caspase inhibitor, C2-ceramide still can induce the release of Cytc and HtrA2 from mitochondria, Can not induce the release of Smac, so that C2-God HT-29 cell apoptosis was induced by amide through the mitochondrial apoptotic pathway. The C2-ceramide-induced release of Cytc and HtrA2 from mitochondria was Caspase-independent while Smac release was Caspase-dependent.