羟基喜树碱诱导肝癌细胞凋亡前后线粒体蛋白质组表达差异的定量分析

A quantitative analysis of mitochondrial protein differential expressions in hydroxycamptothecin-treated hepatoma cells

目的对羟基喜树碱诱导人肝癌细胞SMMC7721凋亡前后的线粒体进行定量蛋白质组学研究。方法用羟基喜树碱诱导肝癌细胞SMMC-7721凋亡，提取线粒体并鉴定其纯度；用可裂解的同位素标记的亲和标签技术标记线粒体蛋白质，利用二维液相色谱-串联质谱分析鉴定蛋白质。结果获得了羟基喜树碱诱导细胞凋亡前后的高纯度线粒体；分析鉴定了细胞凋亡前后，相对表达量差异有统计学意义的蛋白质74种，其中42种线粒体蛋白质在细胞凋亡后的表达量下调，32种蛋白质的表达量在凋亡细胞中上调。这些差异蛋白的分子功能主要表现为能量代谢和核酸的翻译、转录、复制以及细胞骨架等。结论获得了羟基喜树碱诱导肝癌细胞凋亡前后的线粒体差异蛋白信息，将促进对癌细胞凋亡机制及羟基喜树碱药理作用的深入理解，同时，实验所采用的整合技术平台将有助于亚细胞定量蛋白质组学研究。

Objective To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin （HCPT）-treated SMMC-7721 cells by using quantitative proteome. Methods SMMC- 7721 cell apoptosis was induced by HCPT and the mitochondria were isolated with a mitochondria isolation kit. Mitochondrial proteins labeled with a cleavable isotope-ended affinity tag were identified and quantified using two-dimensional liquid chromatography/tandem mass spectrometry. Results Highly purified mitochondria were obtained. Seventy-four mitochondrial proteins, which were statistically significantly altered （P 〈 0.05） in HCPT-treated cells, were identified and analyzed. A total of 42 proteins were significantly downregulated, and 32 were up-regulated in the cells that responded to apoptosis. The functions of these proteins were likely involved in cell energy metabolism, nucleic acid translation and transcription, cytoskeleton, etc. Conclusion Our results about the information of differentially expressed mitochondrial proteins in HCPT- treated cells and the control cells will help to understand the mechanism by which HCPT induces cell apoptosis. The integrated techniques we used in this study will be helpful to the investigation of subeellular quantitative proteomics.