【摘要】 目的了解α干扰素(interferon-α,IFN-α)诱导的抗病毒蛋白MxA和2’,5’-寡腺苷酸合成酶(2’,5’-OAS)在人肝胚瘤细胞株HepG2和HBV稳定转染的HepG2.2.15细胞中的表达情况。方法将培养的HepG2和HepG2.2.15细胞用IFN-α刺激6h,分离细胞总RNA,经逆转录、32P标记后与尼龙膜上的探针微矩阵(macroarray法)进行分子杂交;并以此来分析HepG2和HepG2.2.15细胞IFN抗病毒基因表达谱。用免疫印迹分析IFN抗病毒蛋白;如,MxA、2’,5’-OAS等在肝胚瘤细胞中的表达。结果分析发现在HepG2和HepG2.2.15细胞仅有部分IFN诱导基因表达,多数IFN诱导基因呈低表达或不表达。比较HepG2和HepG2.2.15细胞,发现两者表达IFN诱导基因有差异,即对IFN应答有差异。研究中发现,MxA蛋白在HepG2.2.15细胞中不表达,而在HepG2细胞中却能正常表达。另一种重要的IFN抗病毒蛋白2’,5’-OAS,在HepG2.2.15和HepG2细胞中均能表达;在拉米夫啶处理下,HepG2.2.15细胞甚至比HepG2细胞能更好地表达。结论IFN抗病毒蛋白MxA和2’,5’-OAS在人肝胚瘤细胞株HepG2和HBV稳定转染的HepG2.2.15细胞中表现出差异的表达模式;HBV及其抗原成分影响人肝胚瘤细胞株的IFN抗病毒基因表达。更多还原

【Abstract】 Objective To investigate the expression of IFN antiviral proteins MxA and 2’,5’-oligoadenylate synthetase (2’,5’-OAS) in human hepatoma cell lines, HepG_ 2 and HBV stably-transfected HepG2.2.15 cells after treatment with IFN-α. Methods We treated the HepG_ 2 and HepG2.2.15 cells with IFN-α for 6 h, and then extracted the total cellular RNA for Macroarray analysis. The radio-labelled cDNA was generated from 20 μg of total cellular RNA by reverse transcription at 42℃ for 2 h in the presence of 30 μCi of α-~ 32 P dCTP and reverse transcriptase. Molecular hybridization was performed between the ~ 32 P-labelled cDNA and the Hybond-N~+ membrane arrays. Northern analysis was applied for confirming the expression of some of IFN antiviral proteins MxA and 2’,5’-OAS, etc. Results We found that just partial IFN-inducible genes were expressed in HepG_ 2 and HepG2.2.15 cells. The majority of IFN-inducible genes were lowly responsive or non-responsive to IFN-α treatment. Interestingly, we found that the most important IFN antiviral genes such as MxA, could not express in HepG2.2.15 cells while normally expressed in HepG_ 2 cells. The other important IFN antiviral protein 2’,5’-OAS could express in HepG_ 2 and HepG2.2.15 cells. Further, northern blot analysis confirmed that treatment with 3TC significantly improved the expression of 2’,5’-OAS, but could not up-regulate or restore the expression of MxA in HepG2.2.15 cells. Conclusions There were differential expression models of MxA and 2’,5’-OAS between the HepG_ 2 and HBV stably-transfected HepG2.2.15 cells. The particles and antigens of HBV probably influence the expression of IFN antiviral proteins in human hepatoma cells.更多还原