乙型肝炎病毒X基因转染人胆管癌细胞系及对端粒酶逆转录酶mRNA表达的影响

The effect of hepatitis B virus X gene transfection on expression of human telomerase reverse transcriptase mRNA in human bile duct carcinoma cell lines

目的 了解乙型肝炎病毒X(HBx)基因转染对胆管癌细胞端粒酶逆转录酶 (hTERT)mRNA的表达影响 ,阐明HBx基因调节hTERT基因转录表达在胆管癌发生中的意义。方法 体外培养胆管癌细胞QBC939,应用脂质体介导的基因转染技术 ,将含HBx基因的真核表达载体转染到胆管癌细胞中 ;转染 36h后以增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)表达判定转染成功率 ,流式细胞仪检测转染率 ;收集细胞提取总RNA ,逆转录聚合酶链式反应 (RT PCR)分析转染后细胞内hTERTmRNA的表达变化 ,并通过细胞免疫化学和WesternBlotting了解转染后胆管癌细胞内有无HBx蛋白的表达。结果 转染含HBx基因表达载体和空载体的QBC939转染率为 2 9 6 % ;转染HBx基因后hTERTmRNA表达量比未经转染或转染空载体的hTERTmRNA表达量明显增加 ;细胞免疫组化和WesternBlotting也证实只在转染了HBx基因的胆管癌细胞有HBx蛋白表达。

Objective To study the effect of Hepatitis B virus X(HBx) gene transfection on expression of human telomerase reverse transcriptase (hTERT) mRNA in human bile duct carcinoma cell lines QBC939 and to elucidate the significance of cis-activation of hTERT mRNA by HBx gene on the carcinogenesis of bile duct. Methods QBC939 were cultured in vitro and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein(EGFP)coding sequence using liposome-mediated gene transduction technique. Thirty six hours after transfection,EGFP expression, the indicator of successful transfection in cells, was determined. Flow cytometry was applied to determine the transfection efficiency. Cells were harvested and total RNA was extracted with TRI_(ZOL) Reagent. The expression of hTERT mRNA in QBC939 was assayed by Reverse Transcription Polymerase Chain Reaction. The expression of HBx protein in QBC939 was detected by immunocytochemistry staining and western blotting. Results The transfection efficiency was 29.6% for both HBx expression vector and vector control group. The expression of hTERT mRNA was significantly increased when transfected with HBx expression vector than that transfected with OPTI-MEM medium and vector only. The expression of HBx protein could only be found in the cells when transfected with HBx expression vector by immunocytochemistry staining and western blotting. Conclusion HBx gene transfection may up-regulate the transcriptional expression of hTERT mRNA in bile duct cancinoma cells. The cis-activation of hTERT gene by HBx gene is primary mechanism for carcinogenesis of biliary epithelia after HBV infection.