乙型肝炎病毒X基因对大鼠肾小球系膜细胞增殖和肿瘤坏死因子α表达的影响

HBV X Gene Transfection Induces Glomerular Mesangial Cell Proliferation and Upregulates TNF-α Expression

目的探讨乙型肝炎病毒(HBV)X基因表达的蛋白HBx对体外培养的大鼠肾小球系膜细胞(GMC)表达肿瘤坏死因子α(TNF-α)及细胞增殖的影响。方法扩增HBVX基因,与pCI-neo载体连接,构建pCI-neo-X真核表达载体。采用脂质体法将其转染大鼠GMC,用Western blot法检测HBx的表达;以半定量RT-PCR测定体外培养大鼠GMCTNF-α mRNA的表达;采用ELISA法测定培养上清液中TNF-α的含量;MTT法测定细胞增殖。结果HBx在转染pCI-neo-X的GMC中表达,转染后36、48h HBx表达明显。转染pCI-neo-X组GMC TNF-α mRNA的表达明显高于未转染和空载体转染组。细胞转染pCI-neo-X后培养36、48h,上清液中TNF-α浓度分别为(185.3±70.7)、(140.3±42.1)pg/ml,而未转染组分别为(41.7±10.3)、(43.7±10.6)pg/ml,空载体转染组分别为(44.l±10.1)、(33.3±8.6)pg/ml,均明显低于转染pCI-neo-X组(均P<0.05)。转染pCI-neo-X后可诱导GMC出现增生反应,且在转染后36、48h最明显,与未转染和空载体转染组差异均有显著性意义(P<0.05)。结论HBx可诱导大鼠GMC增生,并在基因和蛋白水平上调GMC TNF-α表达。HBx对GMC的增殖作用可能与其诱导TNF-α的高表达有关。

Objective To study the effect of HBx on glomerular mesangial cell proliferation and TNF-α expression. Methods The HBV X gene was amplified by PCR, inserted into the eukaryotic expression vector pCI-neo, named pCI-neo-X, and confirmed by restrict endonuclease digestion and sequence analysis. PCI-neo-X was then transfected into cultured glomerular mesangial cells. The HBx expression in glomerular mesangial cells was assayed by Western blot and TNF-α mRNA expression by semiquantitative RT-PCR, respectively. TNF-α level in supernatants was measured by ELISA. Mesangial cell proliferation was assayed by MTT. Results The HBx expression was detected in transfected glomerular mesangial cells, and became prominent at 36 and 48 h after transfection. The TNF-α mRNA expression was significantly increased in pCI-neo-X transfection group. TNF-α levels in supernatants in pCI-neo-X transfection group were （185.3±70.7） pg/ml at 36 h and （140.3±42.1） pg/ml at 48 h after transfection, those were （41.7±10.3） pg/ml and （43.7±10.6） pg/ml in control group, and those were （44.1±10.1） pg/ml and （33.3±8.6） pg/ml in vector transfection group, respectively （all P〈0.05）. Glomerular mesangial cell proliferation was induced in pCI-neo-X transfection group and became prominent at 36 and 48 h after transfection. Conelussion HBx can induce glomerular mesangial cell proliferation and increase TNF-α mRNA expression and protein production. Upregulated TNF-α expression may play a role in glomerular mesangial cell proliferation.