乙型肝炎病毒表面抗原小蛋白（SHBs）促进Fas/FasL介导的肝细胞凋亡及机制

Infection of hepatitis B virus (HBV) could effect the Fas/FasL mediated hepatocyte apoptosis, which closely correlated with the clinical courses of hepatits B. It had been well documented that HBx (Hepatitis B virus X protein) and HBc (Hepatitis B virus core protein) could interfere with the Fas/FasL pathway.HBV small surface protein(SHBs) is the most highly expressed protein of HBV,in our previous study, we found that SHBs could significantly enhance anti-Fas CH11-evoked cytotoxicity in HepG2 cells, however, the involved mechanism was far from been elucidated. In the present study,the effects of SHBs on Fas/FasL-mediated apoptosis of hepatocytes was investigated, and the involved mechanism was evaluated, which included the effects of SHBs on the expressions of Fas/FasL, Fas polymerization, formation of DISC, as well as the changes of c-FLIP regulated by PI3K-Akt pathway. This study will ensure the enhancement of SHBs on Fas/FasL-mediated apoptosis of hepatoma cells, and elucidate the involved mechanism, which will be helpful for better understanding of th pathogenicity of HBV.

乙型肝炎病毒（HBV）影响Fas/FasL介导的肝细胞凋亡，与肝炎病程的发生发展密切相关。已有的研究表明HBV编码的HBx蛋白和HBc蛋白均可影响Fas/FasL通路。表面抗原小蛋白SHBs是表达量最高的HBV蛋白，我们先前的研究发现SHBs可促进Fas激动型抗体anti-Fas CH11诱导的HepG2细胞死亡，但相关机制尚不清楚。本研究首先探讨并明确SHBs对Fas/FasL介导肝细胞凋亡的促进作用，在此基础上，进一步从机制上探讨SHBs对Fas/FasL通路各环节的影响，包括Fas/FasL表达量、Fas聚合化程度、死亡诱导信号复合体形成及受PI3K/Akt调控的凋亡抑制因子c-FLIP表达变化等。本研究将明确SHBs对Fas/FasL介导肝细胞凋亡的促进作用及分子机制，所获结果有助于更深入了解HBV的致病机制。

乙型肝炎病毒（HBV）影响Fas/FasL介导的肝细胞凋亡，与肝炎病程的发生发展密切相关。已有的研究表明HBV编码的HBx蛋白和HBc蛋白均可影响Fas/FasL通路。表面抗原（HBsAg/SHBs）是慢性乙型肝炎病毒（HBV）感染患者肝脏和外周血中最为丰富的HBV编码蛋白。我们先前的研究发现HBsAg可促进Fas激动型抗体anti-Fas CH11诱导的HepG2细胞死亡，但相关机制尚不清楚。本研究从进一步深入探讨HBsAg对Fas/FasL介导肝细胞凋亡的影响及机制。本研究通过realtime RT-PCR、半定量RT-PCR和western-blot等方法检测发现HBsAg对p53、mFas、sFas、FasL的表达以及Fas棕榈酰化修饰均无影响；通过免疫沉淀(Immunoprecipitation，IP)等方法发现HBsAg通过促进Fas聚合体的形成，减少DISC上FLIPL/S的募集，从而促进了procaspase-8的活化。我们引入AKT特异性激动剂SC79证明了HBsAg可通过恢复AKT激活所致的Fas受体聚合的减少和procaspase-8切割的减弱，以及FLIPL/S表达的增加，从而逆转由AKT激活所引起的Fas介导肝细胞凋亡的减少。我们还发现，HBsAg通过诱导内质网压力（ER stress）使PDPK1和mTORC2失活进而抑制AKT的磷酸化；HBsAg的L49T，M133I，G145R，S204R和M213I突变导致HBsAg胞内滞留增多，增强内质网压力，可进一步促进Fas介导的肝细胞凋亡。最后，本研究还提示SC79预处理能够缓解anti-Fas Jo2诱导的小鼠ALF，提示使用该类药物在临床上可提高ALF患者生存率。本课题研究阐明了HBsAg对Fas/FasL介导肝细胞凋亡的促进作用及分子机制，这有助于我们更深入了解HBV的致病机制。