目的研究大鼠肝脏卵圆细胞中乙型肝炎病毒编码X蛋白(HBX)对B型转化生长因子(TGFl3)1增殖抑制效应的影响。方法转染HBX基因的大鼠肝脏卵圆细胞系HBX.EGFP.LE/6作为实验组,大鼠肝脏卵圆细胞系LE/6和转染绿色荧光标记空载体的大鼠肝脏卵圆细胞系EGFP.LE/6作为空白对照和阴性对照组。采用real—timePCR和Westernblot分别检测各组TGFl3受体2的mRNA及蛋白表达水平。各组细胞在体外培养中加入5μ/L外源性TGF[M,采用MTT增殖实验比较各组细胞对外源性TGFβ 1细胞增殖抑制效应的不同反应性。结果实验组TGFl3受体2的mRNA和蛋白表达水平较两个对照组均明显下调(P〈0.05)。MTT增殖实验结果表明HBX在体外影响TGFβ1对卵圆细胞的增殖抑制作用,稳定转染HBX的卵圆细胞株HBX—EGFP—LE/6对TGFβI的增殖抑制效应的反应性为18.1%±1.5%,低于其余两组的42.2%±2.8%和41.9%±5.0%(P〈0.05)。结论HBX在肝脏卵圆细胞中的表达影响TGFβ受体2在卵圆细胞中的转录活性和蛋白合成,并且降低肝脏卵圆细胞对TGFβ1增殖抑制效应的敏感度。
[ Abstract] Objective To determine whether hepatitis B virus X (HBX) protein expression affect the oval cells' response to anti-proliferative effect of transforming growth factor β1 ( TGFβ1 ) in oval cells. Methods Real-time PCR, Western blot analysis were performed to detect the expression of TGFβR lI in HBX-transfected oval cells named HBX-EGFP-LE/6, and EGFP-LE/6, LE/6 control cells. In addition, exogenous TGFβ1 was added into all three oval cell lines, MTI assay was preformed to clarify different responses to the anti-proliferative effect of TGFβ1. Results The TGFI3R II mRNA levels in LE/6 and EGFP-LE/6 cells were ( 10. 2 ± 1.8 ) and ( 8.8 ± 0. 9 ) folds of those in HBX-EGFP-LE/6 cells, the difference was significant (P 〈 0. 05 ). HBX protein expression also reduced the protein levels of TGFβR lI in HBX-EGFP-LE/6 oval cells, compared to the control cells. The MTT results exhibited that, after TGFIS1 addition, proliferative inhibition rate in the HBX-EGFP-LE/6 cells was 18.1% ± 1.5% while those in control cells were 42. 2% ± 2. 8% and 41.9% ± 5.0% , the difference was significant ( P 〈 0. O1 ). Conclusion HBX protein expression affects TGFβR I1 transcriptional activity and protein synthesis, and insensitive oval cells to anti-proliferative effect of TGFβ1.