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目的探讨乙型肝炎病毒x蛋白(HBx)能否诱导肝前体细胞(HPCs)发生上皮-间质转化(EMT)。方法将重组质粒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,经稻瘟菌素(blasticidin)筛选,获得稳定表达HBx的HP14.5细胞。观察其形态变化,通过实时定量PCR(real time-PCR)及Western blot法检测其间质细胞标志物和上皮标志物在mRNA和蛋白水平的表达量,并采用划痕实验检测其细胞迁移能力的变化。结果 HP14.5细胞稳定表达HBx蛋白后细胞由多角形变为长梭形;与对照组相比,神经钙黏素(N-cadherin)、Snail、vimentin的mRNA和蛋白水平表达均升高,具有统计学差异(P<0.05)而上皮性钙黏素(E-cadherin)、细胞角蛋白18(CK18)的mRNA和蛋白表达量显著降低(P<0.05;同时其迁移能力显著升高(P<0.05)。结论 HBx能诱导肝前体细胞发生EMT,并增强其迁移能力。
Objective To investigate whether hepatitis B virus x protein (HBx) can induce epithelial-mesenchymal transition (EMT) in hepatic precursor cells (HPCs). Methods Recombinant plasmid pSEB-Flag-HBX and packaging plasmid pAmpho were co-transfected into human embryonic kidney epithelial cell line 293T and packaged to obtain recombinant retrovirus carrying HBx gene and infected with mouse hepatic precursor cell HP14.5. Blasticidin screening to obtain HP14.5 cells stably expressing HBx. Morphological changes were observed. Real-time PCR and Western blot were used to detect the mRNA and protein expression levels of mesenchymal cell markers and epithelial markers. Scratch assay was used to detect the cell migration ability Variety. Results The expression of HBx protein in HP14.5 cells changed from polygons to fusiform cells. Compared with the control group, the mRNA and protein levels of N-cadherin, Snail and vimentin increased, with statistics (P <0.05), while the mRNA and protein expressions of E-cadherin and CK18 were significantly decreased (P <0.05; meanwhile, the migration ability was significantly increased (P <0.05 ) Conclusion HBx can induce EMT in liver precursor cells and enhance its migration ability.