乙肝病毒感染对细胞基本自噬的影响

【目的】慢性乙肝病毒(Hepatitis B virus,HBV)感染在肝硬化和肝癌的发生过程中起着重要的作用,通过研究HBV感染对细胞基本自噬的影响,为HBV感染诱发肝癌以及HBV的免疫逃逸机理研究提供新的思路。【方法】本研究利用乙肝病毒表达质粒瞬时或稳定转染不同肝细胞,通过计数绿色荧光蛋白(greenfluorescent protein,GFP)聚集数目检测自噬小体形成,western blot检测LC3(microtubule-associated proteinlight chain 3,微管相关蛋白质轻链3)脂酰化和p62的降解,通过构建HBV B型和C型X蛋白(HBx)的表达质粒并瞬时转染肝癌细胞和正常肝细胞,对不同基因型X蛋白对细胞自噬的影响进行了分析。【结果】乙肝病毒感染后促进了LC3的脂酰化和p62的降解,增加了自噬小体的形成,增强了细胞的基本自噬。进一步研究发现,HBV感染增强的细胞基本自噬水平由HBx所引发,且C型HBx比B型对细胞基本自噬的增加更加显著。【结论】HBV通过HBx增强细胞的基本自噬,且不同基因型HBx对细胞基本自噬的增强程度不同,为进一步阐明HBV感染机理奠定了基础。

Infection with Hepatitis B virus enhances basal autophagy

Abstract: Objective] Hepatitis B virus (HBV) is a major human pathogen that chronically infects 400 million people worldwide. Chronic infection of HBV plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). To clarify the mechanism of HBV-related HCC and immune escape of HBV, we investigated the relationship between infection of HBV and basal autophagy. Methods] To examine the number of autophagosomes upon transfection with HBV, HepG2.2.15 cells were transfected with green fluorescent protein-microtubule-associated protein 3(GFP-LC3) and observed by fluorescence microscopy. Huh7 or HepG2 cells was transiently transfected with HBV expression vector pHBV1.3, then the phosphatidylethanol -amine conjugation of microtubule-associated protein 3 (LC3) and the degradation of p62, both of which are specific indictors of autophagy, were evaluated by western blot. Moreover, HepG2 or Chang liver cells were transiently transfected with the constructed HBV X protein(HBx) expression vector in order to evaluate autophagic status of the cells. Results] HBV and HBx were both able to increase the autophagosomes formation as well as the enhancement of autophagic flux. Notably, C type HBx had a more increment of autophagy than B type does. Conclusion] HBV can enhance basal autophagy and the increment is dependent on HBx. Different genotypes of HBx had different effects on basal autophagy. Take together, these findings will help us to clarify the mechanism of HBV infection and the development of Hepatitis B to HCC in the future study.