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目的 探讨干扰素γ(IFN γ)和肿瘤坏死因子α(TNF α)对乙型肝炎病毒转录后调节机制的影响。方法 在体外构建含有乙型肝炎病毒转录后调节序列 (HPRE)片段的pDM1 38质粒 (含有CAT报告基因 ) ,应用脂质体介导方法转染HepG2 细胞 ,观察IFN γ和TNF α对重组质粒载体CAT报告基因活性表达的影响 ,CAT基因活性的检测采用ELISA检测试剂盒。结果 成功构建了含有HPRE片段的重组质粒载体。转染重组质粒的HepG2 细胞CAT基因活性表达明显增强 ,而转染空载体的HepG2 细胞仅有本底表达 ,分别为 896pg ml± 2 1 4pg ml和 37pg ml± 1 1pg ml。IFN γ和TNF α对转染重组质粒的HepG2 细胞CAT活性表达的影响均具有浓度依赖性 ,而对空载体CAT活性的表达无影响。转染重组质粒的HepG2 细胞在未加入细胞因子时 ,其CAT表达活性为 896pg ml± 2 1 4pg ml;与IFN γ、TNF α及IFN γ +TNF α孵育后 ,其CAT表达活性分别为 32 4pg ml± 57pg ml,396pg ml± 82pg ml,1 75pg ml± 36pg ml,与未加入时比较 ,t=5 1 9,4 39,6 68,P <0 0 1 ,差异有显著意义。结论 IFN γ和TNF α可能通过抑制HPRE的功能 ,调控乙型肝炎病毒基因的表达
Objective To investigate the effects of interferon γ (IFN γ) and tumor necrosis factor α (TNF α) on the post-transcriptional regulation of hepatitis B virus (HBV). Methods Plasmid pDM1 38 (containing the CAT reporter gene) containing the post-transcriptional regulation sequence (HPRE) of hepatitis B virus was constructed in vitro and transfected into HepG2 cells by liposome-mediated method. The effect of IFNγ and TNFα on the expression of recombinant plasmid vector CAT reporter gene activity, CAT activity was detected using ELISA kit. Results The recombinant plasmid vector containing HPRE fragment was successfully constructed. The expression of CAT gene in HepG2 cells transfected with the recombinant plasmids was significantly enhanced, whereas the expression of HepG2 cells transfected with empty vector was only 896 pg ml ± 214 pg ml and 37 pg ml ± 1 pg ml, respectively. The effects of IFNγ and TNFα on the CAT activity of HepG2 cells transfected with recombinant plasmids were concentration-dependent, but had no effect on the expression of CAT. The CAT expression activity of HepG2 cells transfected with recombinant plasmids was 896pg ml ± 214pg ml when no cytokines were added. CAT activity of HepG2 cells transfected with recombinant plasmids was 32 4pg ml ± 57 pg ml, 396 pg ml ± 82 pg ml, 1 75 pg ± 36 pg ml, t = 5 1 9, 4 39, 688, P 0 01, respectively, with significant difference. Conclusion IFN γ and TNF α may regulate the expression of hepatitis B virus gene by inhibiting the function of HPRE