乙型肝炎病毒X蛋白对肝细胞L02增殖及GSK3β表达的影响

Effects of hepatitis B virus X protein on cell proliferation and GSK3β expression of human liver cell line L02

目的:通过研究乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)对人肝细胞株L02细胞增殖、细胞周期以及细胞中糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK3β)表达的影响,探讨乙型肝炎病毒(hepatitis B virus,HBV)相关性原发性肝细胞癌(hepatoccellular carcinoma,HCC)的发生机制。方法:用HBx腺病毒(Ad-HBx)感染人肝细胞株L02细胞后,RT-PCR法检测L02细胞中HBx和GSK3β mRNA表达情况;MTT法检测L02细胞的增殖率变化;FCM法检测细胞周期中各时相所占比例;蛋白质印迹法检测HBx、总GSK3β(total-GSK3β,t-GSK3β)、磷酸化GSK3β(phospho-GSK3β,p-GSK3β)、β-连环素(β-catenin)以及细胞用期蛋白(cyclinD1)等蛋白的表达水平。结果:Ad-HBx感染L02细胞后,HBx的mRNA和蛋白均出现表达,细胞增殖率随着时间的延长而增加;G1期细胞所占比例较对照组减少,S期和G2期细胞比例较对照组增加(P<0.05)。感染Ad-HBx后,t-GSK3β在mRNA和蛋白水平上均无明显变化,而p-GSK3β、β-catenin以及cyclinD1蛋白的表达量增加(P<0.05)。结论:HBx可能通过促进人肝细胞株L02细胞中GSK3β的磷酸化,激活Wnt/β-catenin下游信号通路,从而促进细胞的增殖。

Objective：To investigate the effects of hepatitis B virus（HBV） X protein（HBx） on cell proliferation,cell cycle and the glycogen synthase kinase 3β（GSK3β） expression of human liver cell line L02,and discuss the mechanism of carcinogenesis of HBV-associated hepatocellular carcinoma（HCC）.Methods：The human liver cell line L02 was infected with Ad-GFP or Ad-HBx.The expressions of HBx and GSK3β mRNAs were identified by RT-PCR.MTT method and flow cytometry were used to detect the cell proliferation and cell cycle distribution,respectively.The expression levels of HBx,total-GSK3β（t-GSK3β）,phospho-GSK3β（p-GSK3β）,β-catenin and cyclin D1 proteins were examined by Western blotting.Results：The positive expressions of HBX mRNA and protein in L02 cells were detected after infection with Ad-HBx.The cell proliferation rate was elevated in a time-dependant manner.The percentage of Ad-HBx-infected cells at G1 period was lower than that in the control group,while the percentages of Ad-HBx-infected cells at S and G2 periods were both higher than those in the control group（P0.05）.The expression levels of t-GSK3β mRNA and protein in Ad-HBx-infected cells had no significant changes,and the expression levels of p-GSK3β,β-catenin and cyclin D1 proteins were increased compared with those in the control group（P0.05）.Conclusion：HBx may participate in the activation of downstream Wnt/β-catenin signal pathway through promoting the phosphorylation of GSK3β in L02 cells,and eventually increase the cell proliferation ability.