人颗粒溶素对肝癌细胞SMMC-7721凋亡、线粒体跨膜电位及细胞色素C释放的影响

Effect of human granulysin on apoptosis, mitochondrial transmembrane potential and cytochrome C release of SMMC-7721 cells

目的构建携带人颗粒溶素（GLS）的真核表达质粒pBudCE4．1／GLS并研究其表达后对肝癌细胞SMMC-7721凋亡，线粒体跨膜电位与细胞色素C转位的影响。方法将RT—PCR扩增出的人GLS编码基因克隆入pBudCE4．1载体中构建真核表达质粒pBudCE4．1／GLS并将其转染肝癌细胞株SMMC一7721，用RTPCR、免疫细胞化学检测GLS的表达；用Hoechst法及电镜检测细胞的凋亡状况；用荧光染料Mitocapture^TM检测线粒体跨膜电位、用Western blot法检测细胞色素C从线粒体的释放。结果成功构建了携带GLS的真核表达质粒pBudCE4．1／GLS；在转染的肝癌细胞株SMMC-7721中，从转录和翻译水平都检测到了目标基因GLS的表达产物；重组质粒转染SMMC-7721细胞后，细胞核染色质固缩，呈致密浓染的凋亡状态，线粒体膜跨电位下降并伴随有细胞色素C从线粒体至细胞质的释放。结论携带人GLS的真核表达质粒在肝癌细胞SMMC-7721中表达后有致细胞凋亡作用，引起线粒体跨膜电位下降与细胞色素C的释放可能是其诱导肿瘤细胞凋亡的途径之一。

Objective To construct a plasmid carrying granulysin （GLS） and to study the effect of the GLS on apoptosis, mitochondrial transmembrane potential and cytochrome C release of SMMC-7721 cells. Methods The coding sequence of the GLS was amplified from the total RNA of human CTL cells, and it was inserted into pBudCE4.1 plasmid and then it was used to transfect SMMC-7721 cells. The expression of GLS was detected by RT-PCR and confirmed by immunocytochemistry method. Cell apoptosis was ascertained by Hoechst staining and electron microscopy; mitochondrial transmembrane potential was detected using Mitocapture^TM and cytochrome C release was studied using Western blot. Results Recombinant pBudCE4.1/GLS plasmid was successfully constructed. GLS protein was successfully expressed in the SMMC- 7721 cells and it induced apoptosis of the SMMC-7721 cells, and at the same time, mitochondrial transmembrane potential was reduced and cytoehrome C was released from mitochondria into the cytosol. Conclusions GLS gene carried by recombinant plasmid could express in SMMC-7721 cells and induce cells apoptosis. The change of mitochondrial transmembrane potential and the release of cytochrome C might be one of the key factors of apoptosis induced by GLS.