摘要
目的构建KM小鼠HBx (hepatitis B virus X)表达模型,探讨HBx对正常免疫功能小鼠肝脏细胞凋亡和周期的影响及其机制。方法采用qRT-PCR和Western blot检测HBx在肝前体细胞(hepatic progenitor cells, HPCs)中的表达;将表达HBx的肝前体细胞通过肝门静脉注射到KM小鼠体内建立动物模型,手术120 d后分别使用qRT-PCR,Western blot和免疫组化的方法检测小鼠肝脏组织中HBX的表达;Western blot和qRT-PCR方法检测小鼠肝脏细胞凋亡和周期相关因子以及Akt/mTOR信号通路因子的表达水平;采用TUNEL-FITC/DAPI方法检测小鼠肝脏细胞的凋亡率。结果 HBx组的小鼠肝脏细胞的凋亡率降低,促凋亡因子caspase-9和Bad的mRNA和蛋白质表达水平均降低;CDK2、cyclinD1的mRNA表达水平升高,CDK2、cyclinD1、p-Akt以及p-mTOR的蛋白质表达水平升高;给予携带HBx的小鼠Akt磷酸化抑制剂MK2206可显著扭转HBx对上述基因的mRNA及蛋白质的表达影响。结论 HBx在体内激活Akt/mTOR信号通路抑制了小鼠肝脏细胞的凋亡和上调细胞周期调控因子的表达。
Objective To investigate the effect of hepatitis B virus X(HBx)on apoptosis and cell cycle of hepatocytes in mice with normal immune functions and explore the underlying mechanisms.Methods We transduced HBx gene in cultured mouse hepatic progenitor cells(HPCs)via lentiviral vectors to obtain HBx-expressing mouse HPCs(HBx-EGFP-14-19 cells),and the expression of HBx at both mRNA and protein levels in the cells were examined using qRT-PCR and Western blotting.HBx-EGFP-14-19 cells were then injected into the hepatic portal vein of KM mice,and 120 d after the injection,the liver tissues of the mice were collected to examine the expression of HBx using qRT-PCR,Western blotting and immunohistochemistry.The apoptotic index of the hepatocytes of the mice was investigated using TUNEL-FITC/DAPI method,and the expression levels of apoptosis-related and cell cycle-related factors were detected using qRT-PCR and Western blotting.We also detected the changes in the expression levels of the molecules associated with Akt/mTOR signaling in response to an Akt inhibitor in the mice with HBx-EGFP-14-19 cell injection.Results We successfully obtained HBx-EGFP-14-19 cells that expressed HBx mRNA and protein.At 120 d after injection of HBx-EGFP-14-19 cells,the hepatocytes of the mice showed a decreased apoptotic index,lowered expression of the pro-apoptotic factors caspase-9 and Bad,and increased mRNA expression of CDK2 and cyclin D1 and protein expression of CDK2,cyclin D1,p-Akt and p-mTOR.Treatment of the mice with the Akt inhibitor MK2206 obviously abolished these changes induced by HBx overexpression.Conclusion HBx suppresses apoptosis and accelerates cell cycle progression of the hepatocytes in mice by activating Akt/mTOR signaling pathway.
作者
王薛
霍本念
刘洁
黄欣
张思遥
冯涛
WANG Xue;HUO Bennian;LIU Jie;HUANG Xin;ZHANG Siyao;FENG Tao(Molecular Medicine and Cancer Research Center,Department of Biochemistry and Molecular Biology,College of Basic Medical Sciences,Chongqing Medical University,Chongqing,400016,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2019年第10期931-938,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81071770)~~