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目的构建稳定表达乙型肝炎病毒X蛋白(HBx)的肝前体细胞株,检测对肝前体细胞增殖,细胞周期及Wnt/β-catenin信号通路的影响。方法重组质粒pSEB-Flag-HBx与包装质粒pAmpho共同转染人胚胎肾上皮细胞系293T细胞,包装获得携带HBx基因的重组逆转录病毒,并感染小鼠肝前体细胞HP14.5,稻瘟菌素(blasticidin)筛选出稳定表达HBx基因的细胞克隆并扩大培养(HP14.5/HBx组),设HP14.5/Rv(空质粒pSEEB-Flag感染组)及空细胞组HP14.5组为对照组。MTS比色法检测各组细胞的增殖活力,流式细胞术检测细胞周期中各时相所占的比例,荧光定量PCR检测cyclin D1和c-myc mRNA的表达量,Western blot法检测HBx、GSK3β、p-GSK3β(ser-9)、β-catenin、cyclin D1以及c-myc等蛋白的表达。结果 RT-PCR和Westernblot法检测到HBx基因和蛋白阳性表达,与对照组相比,HP14.5/HBx细胞增殖活力显著增加(P<0.05);G1细胞比例明显减少(P<0.05),S期和G2细胞比例显著升高(P<0.05);cyclin D1和c-myc mRNA的表达量显著升高(P<0.05);GSK3β蛋白水平表达无明显变化(P>0.05),p-GSK3β、β-catenin、cyclin D1和c-myc蛋白的表达水平显著升高(P<0.05)。结论成功筛选出稳定表达HBx的肝前体细胞株,HBx通过活化Wnt/β-catenin信号途径促进肝前体细胞的增殖。
Objective To construct a hepatoblastoma cell line stably expressing Hepatitis B virus X protein (HBx) and detect the effect on the proliferation, cell cycle and Wnt / β-catenin signaling pathway of hepatic precursor cells. Methods Recombinant plasmid pSEB-Flag-HBx was co-transfected with 293T cell line of human embryonic kidney epithelial cells with the packaging plasmid pAmpho to obtain a recombinant retrovirus carrying the HBx gene and infected with mouse hepatic precursor cells HP14.5, (Blasticidin) were selected to clone and expand the cell clones stably expressing HBx gene (HP14.5 / HBx group). HP14.5 / Rv (empty plasmid pSEEB-Flag infection group) and empty cell group HP14.5 group were used as control group. MTS colorimetric assay was used to detect the proliferative activity of each group. Flow cytometry was used to detect the proportion of each phase in the cell cycle. The expression of cyclin D1 and c-myc mRNA was detected by real-time PCR. The expression of HBx, GSK3β , P-GSK3β (ser-9), β-catenin, cyclin D1 and c-myc protein expression. Results The expression of HBx gene and protein was detected by RT-PCR and Western blot. Compared with the control group, the proliferation activity of HP14.5 / HBx cells was significantly increased (P <0.05), while the proportion of G1 cells was significantly decreased (P <0.05) (P <0.05); the expression of cyclin D1 and c-myc mRNA increased significantly (P <0.05); the expression of GSK3β protein had no significant change (P> 0.05) The expression of β-catenin, cyclin D1 and c-myc protein were significantly increased (P <0.05). Conclusion HBx-positive hepatocyte precursor cells were successfully selected and HBx could promote the proliferation of hepatic precursor cells by activating the Wnt / β-catenin signaling pathway.