摘要
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 establish HP14.5 cell line stably expressing hepatitis B virus X （HBx） and detect the effect of HBx on cell proliferation, cell cycle and the wnVI3-catenin signal pathway of hepatic progenitor cells. Methods The plasmid of pSEB-Flag-HBx and the packaging plasmid pAmpho were co-transfected into HEK 293T cells to construct the recombinant retrovirus carrying HBx gene. The recombinant retrovirus wan then transfected into mouse hepatic progenitor cells HP14.5. The blasticidin-resistant clones of HBx cells （HP14.5/HBx） were selected out and cultured as the experimental group, paral- leled with the vector control HP14.5/Rv （HP14.5/pSEEB-Flag） and HP14.5 cell as negative control. The cell proliferation was tested by MTS assay, the cell cycle was measured by flow cytometry, the expression levels of cyclin D1 and c-myc mR- NA were tested by real time PCR, and the expression levels of HBx, cyclin D1, c-myc, GSK3β, p-GSK315 （ser-9）, β-catenin proteins were examined by Western blotting. Results The expression of HBx at both mRNA and protein levels was positive in HP14.5/HBx cell line as confirmed by RT-PCR and Western blotting. Compared with the control groups, the proliferation of HPI4.5/HBx cells increased significantly （P〈O. 05）, and the expression level of cyclin D1 and c-myc mRNA rose signifi- cantly （ P 〈 0.05）. Gl-phase cell proportion was reduced while the proportion in S and G2 phases went up significantly （ P 〈 0.05）. The expression levels of cyclin D1, c-myc, β-catenin and p-GSK313 （ser-9） proteins increased significantly （P 〈 0.05） except GSK315 （P 〉 0.0.5 ）. Conclusion The hepatic stem cell line stably expressing HBx has been constructed successfully. HBx can promote the proliferation and malignant transformation of hepatic stem cells via the activation of Wnt/ β-catenin signal pathway.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2013年第3期256-260,共5页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81071770
81201679)
