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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 virus X protein (HBx) on the expression and localization of β-catenin in Hepatic progenitor (HP) 14-19 cells. The mechanism of HCC is provided experimental evidence. Methods HP 14-19 cells were infected with recombinant adenovirus Ad-GFP-HBx and Ad-GFP respectively. The transcription of HBx gene mRNA was detected by RT-RCR. The mRNA level of β-catenin was detected by Real-time PCR. The expression of HBx, total-glycogen synthase kinase 3β (t-GSK3β), phospho-GSK3β (p-GSK3β) and β-catenin protein were detected by immunohistochemical method. catenin distribution. Results Both recombinant adenovirus Ad-GFP-HBx and Ad-GFP could efficiently infect HP 14-19 cells with an infection efficiency of 90%. In recombinant adenovirus Ad-GFP-HBx infected HP 14-19 cells, Compared with Ad-GFP infection group, the relative expression of t-GSK3β protein in Ad-GFP-HBx infection group had no significant change (P> 0.05), but the relative expression of p-GSK3β protein (P <0.05). The expression of β-catenin at the gene and protein levels was significantly increased (P <0.05), and it accumulated in the cytoplasm with a tendency of translocation to the nucleus. Conclusion HBx can promote the phosphorylation of GSK3β in HP 14-19 cells and inhibit the degradation of β-catenin, leading to a large accumulation of β-catenin in the cytoplasm and its transfer to the nucleus. Activation of the canonical Wnt signaling pathway in hepatic precursor cells by HBx may be the molecular basis leading to the malignant transformation of liver precursor cells.