【摘要】 对羟基喜树碱诱导肝癌细胞凋亡前后的细胞核蛋白质组进行定量研究,获得凋亡前后细胞核蛋白质组中差异蛋白表达量相对变化的信息,为在亚细胞定量蛋白质组水平上深入探讨羟基喜树碱的作用机理提供实验依据。分离提取羟基喜树碱诱导肝癌细胞凋亡前后的细胞核并鉴定其纯度,用含稳定同位素的化学试剂c-ICAT标记细胞凋亡前后的细胞核蛋白,对细胞核标记蛋白进行消化和纯化,利用基于多维色谱-线性离子阱/静电场轨道阱质谱联用技术的鸟枪(Shotgun)法策略及c-ICAT定量策略分析鉴定蛋白质,获得同一种肝癌细胞核蛋白质在凋亡前后细胞中的表达量变化比值。分析鉴定了在细胞凋亡前后的表达量差异有显著统计学意义(P<0.05)的肝癌细胞核蛋白42个,其中,12个蛋白的表达量在羟基喜树碱诱导细胞凋亡后下调,30个蛋白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与细胞增殖、凋亡和分化、能量代谢、核酸代谢以及细胞骨架相关。更多还原

【Abstract】 The experimental foundation for investigating the pharmacological action of hydroxycamptothecin at subcellular quantitative proteomic level can be obtained depending on the information of differentially expressed nuclear proteins in hydroxycamptothecin-treated cells and control cells. The apoptosis was induced by hydroxycamptothecin in hepatoma cells, the nucleus of cells were isolated and verified with western blot. Nuclear proteins labelled with cleavable isotope-coded affinity tag (c-ICAT) reagent were digested and purified. The expression ratio of the identical nuclear protein derived from apoptosis cell and control cell can be gained using shotgun proteomic method based on multiple dimensional liquid chromatography-linear ion trap/orbitrap mass spectrometer combined with c-ICAT strategy. A total of 42 nuclear proteins were significantly (P<0.05) altered in hydroxycamptothecin-treated cells, among them,12 proteins showed significantly down-regulation, and 30 proteins showed up-regulation compared with control cells. The function of these proteins were likely involved in life processes of cells such as proliferation, apoptosis, differentiation, energy metabolism, nucleic acid synthesis and metabolism,structure of cell skeleton.更多还原