乙肝病毒X蛋白诱导肝癌细胞凋亡的信号转导途径

乙型肝炎病毒(hepatitisBvirus,HBV)X蛋白(HBX)与肝癌(hepatocellularcarcinoma,HCC)的发生具有密切的关系.HBX不但具有拮抗细胞凋亡的作用,还具有促进细胞凋亡的作用.为了进一步探讨HBX促细胞凋亡作用的分子机制,通过脂质体转染的方法将携带X基因的真核表达载体pCMVX导入H7402肝癌细胞,使乙肝病毒x基因(HBx)瞬时过量表达.流式细胞仪检测结果显示,在瞬时转染3μgpCMVX质粒后,肝癌细胞发生凋亡.为阐明HBX诱导细胞凋亡的信号转导途径,对HBX与线粒体释放细胞色素c的关系做了初步探讨.通过罗丹明123染色,经流式细胞仪分析,显示在转染HBx基因后细胞线粒体膜电位明显下降,表明HBX可促进细胞色素c从线粒体释放增加.Western印迹检测结果显示,肝癌细胞在导入HBx基因后,细胞凋亡线粒体转导途径中细胞色素c、Apaf1、procaspase3和procaspase9等的表达水平均上调.研究结果说明,HBX可通过影响线粒体凋亡途径促进肝癌细胞凋亡.

Signaling Pathway of Apoptosis Induced byHepatitis B Virus X Protein

HBX plays an important role in both pro-apoptosis and anti-apoptosis.To identify the molecular mechanism underlying the pro-apoptotic effect, plasmid pCMV-X harboring HBX cDNA was constructed succedssfully,thereafter was transiently transfected into liver cancer cell line H7402 by lipofectamine.RT-PCR and Western blot analysis found that HBX gene was overexpressed at mRNA as well as protein level after 48 hours transfection.Flow cytometry analysis showed that transfection with doses of 1 μg, 2 μg and 3 μg of plasmid led to dramatically increase in apoptotic rate in dose dependent manner,compared with empty vector.Furthermore,to illustrate the relative factors of apoptosis induced by HBX expression, we investigated the relationship between HBX and release of cytochrome c from mitochondria. After transfection rhodamine 123 staining was employed to examine the membrane potential change of mitochondria by flow cytometry analysis. It was showed that the membrane potential of mitochondria decreased, suggesting that HBX protein promoted the release of cytochrome c from mitochondria in the hepatoma cells. The expression level of the factors involved in mitochondria, including cytochrome c, Apaf-1, pro-caspase 3 and pro-caspase 9, was up-regulated in the apoptotic cells in different degree. The results illuminated that HBX protein promoted apoptosis in hepatocellular carcinoma through mitochondria signal pathway.