摘要
目的探讨乙肝病毒X蛋白(Hepatitis B Virus X,protein,HBx)对肝前体(Hepatic progenitor,HP)14-19细胞中β-连环素(β-catenin)表达及定位的影响,为研究HBV相关性肝癌的发生机制提供实验依据。方法分别用重组腺病毒Ad-GFP-HBx和Ad-GFP感染HP 14-19细胞,RT-RCR法检测HBx基因mRNA的转录;Real-time PCR法检测β-catenin基因mRNA的水平;Western blot法检测HBx、总糖原合成酶激酶3β(total-glycogen synthase kinase 3β,t-GSK3β)、磷酸化GSK3β(Phospho-GSK3β,p-GSK3β)及β-catenin蛋白的表达;免疫细胞化学法检测β-catenin的分布情况。结果重组腺病毒Ad-GFP-HBx和Ad-GFP均能高效感染HP 14-19细胞,感染效率均可达到90%;在重组腺病毒Ad-GFP-HBx感染的HP 14-19细胞中可检测到HBx基因mRNA的转录和蛋白的表达;与Ad-GFP感染组相比,Ad-GFP-HBx感染组t-GSK3β蛋白相对表达量无明显变化(P>0.05),但p-GSK3β蛋白相对表达量增加(P<0.05);β-catenin在基因和蛋白水平上表达明显增高(P<0.05),并在胞质中大量积聚,且有向胞核转位的趋势。结论 HBx可通过促进HP 14-19细胞中GSK3β磷酸化,抑制β-catenin的降解,从而导致β-catenin在胞质中大量积聚并向核内转移。HBx对肝前体细胞中经典Wnt信号通路的激活,可能是导致肝前体细胞恶性转化的分子基础。
Objective To investigate the effect of hepatitis B X protein（HBx）on expression and location of β-catenin in mouse hepatic progenitor cells（HP 14-19）.Methods HP 14-19 cells were infected with recombinant adenovirus AdGFP-HBx and Ad-GFP respectively,and determined for transcription of HBx mRNA by RT-PCR,for β-catenin mRNA level by real-time PCR,for expressions of HBx,total-GSK3β（t-GSK3β）,phosphor-GSK3β（p-GSK3β）and β-catenin by Western blot,and for distribution of β-catenin by immunocytochemical assay.Results Both the infection efficacies of Ad-GFP-HBx and Ad-GFP to HP 14-19 cells reached 90%.The transcription of HBx mRNA and expression was detected in HP 14-19 cells infected with Ad-GFP-HBx.As compared with that in HP 14-19 cells infected with Ad-GFP,the expression level of t-GSK3β in HP 14-19 cells infected with Ad-GFP-HBx showed no significant difference（P 0.05）, while that of p-GSK3β increased（P 0.05）.Meanwhile,the expression of β-catenin in the cells infected with Ad-GFPHBx increased at both mRNA and protein levels（P 0.05）.A large quantity of expressed β-catenin were accumulated in cytoplasm and showed a tendency of transferring into cell nucleus.Conclusion HBx promoted the phosphorylation of GSK3β and inhibited the degradation of β-catenin,resulting the accumulation of a large quantity of β-catenin in cytoplasm and their transferring to cell nucleus.The activation of classic Wnt signaling pathway may be a molecular basis of malignant transformation of hepatic progenitor cells.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第5期603-607,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(81071770)
青年科学基金项目(81201679)
关键词
乙型肝炎病毒X蛋白
Β-连环素
肝前体细胞
肝癌
WNT信号通路
Hepatitis B Virus X protein（HBx）
β-catenin
Hepatic progenitor cells
Hepatocellular carcinoma
Wnt signaling pathway
