PIG11高表达诱导HepG2细胞凋亡及其机制的初步探讨

目的利用已建立的稳定高表达PIG11蛋白的HepG2细胞株,探讨PIG11蛋白表达对HepG2细胞凋亡的影响,以及线粒体膜电位改变和Cyt C释放在凋亡过程中的作用。阐明PIG11诱导细胞凋亡的分子机制。方法 PI染色,流式检测细胞的凋亡情况。用Rh123标记,流式测定线粒体膜电位。Western Blot检测胞浆和线粒体中Cyt C的含量变化。结果流式细胞仪检测Rh123荧光强度,结果显示：pLXSN-PIG11-HepG2细胞荧光强度（5.4±0.15）低于HepG2细胞（14.7±0.56）和pLXSN-HepG2细胞（13.2±0.53）（P〈0.01）。表明PIG11高表达诱导细胞凋亡过程中存在线粒体膜电位去极化。用CsA（10μmol/L）抑制各组细胞的线粒体通透性转移孔后,流式凋亡检测结果显示pLXSN-PIG11-HepG2细胞凋亡率明显降低。Western Blot检测发现存在线粒体Cyt C向胞浆释放。结论 PIG11高表达可诱导HepG2细胞凋亡,PIG11诱导细胞凋亡机制可能由线粒体膜电位改变及Cyt C向胞浆释放而介导。

The Apoptosis Induced by PIG11 Protein in HepG2 Cells and the Discussion of its Mechanism

Objective Explore the effect of PIG11 gene expression on HepG2 cell line apoptosis with HepG2 cells line high expressing PIG11 gene.Mitochondrial dysfunction were researched to get the more information of apoptotic mechanism induced by PIG11.Methods The role of PIG11 in apoptosis was analyzed by the flow cytometer scans（FACS）.Apoptotic cells were quantified by sub-G1DNA content.The fluorescent probe rhodamine 123 was used to analyze the mitochondrial transmembrane potential（Δψm） and the cellular fluorescence intensity was measured by FACS.Cytochrome C（Cyt C） protein was analyzed by western blot.Results Rh 123 fluorescence showed a decrease in pLXSN-PIG11-HepG2 cells（5.4±0.15）,compared with HepG2 cells（14.7±0.56）and pLXSN-HepG2 cells（13.2±0.53）（P0.01）,suggesting a loss of the Δψm in pLXSN-PIG11-HepG2 cells.The apoptosis was blocked visibly by CsA（an effective and specific inhibitor of mitochondrial permeability transition pore,mtPTP）.Cyt c content extracted from HepG2 cells with pLXSN-PIG11 was decreased considerably in the mitochondria,whereas it increased in the cytosol.These demonstrated that loss of Δψm with the release of Cyt c in the apoptosis induced by PIG11.Conclusion These findings suggest that over-expression of PIG11 induce HepG2 cells apoptosis through mitochondrial pathway.