乙型肝炎病毒影响肝细胞对双链DNA诱导产生的Ⅰ型干扰素应答

摘要

Objective: To explore the possibility that double-stranded DNA ( dsDNA ) may trigger the type I interferon production and the influence of hepatitis B virus on this response in hepatocyte-derived cells. Methods; A hepatoma cell line HepG2 and its derivate with HBV, HepG2.2. 15, were treated with poly( dA-dT), a synthetic double-stranded DNA. Cellular total RNAs were extracted and detected for IFN-β, IFIT1, and TNF-α expression by real time RT-PCR. The translocation of IRF3 and NF-KB and the phosphorylation of TBK1 were detected by Western blot. HepG2 cells were transfected with HBV replicative plasmid of HBV1.3, or HepG2215 cells were transfected with small interfering RNA of HBV ( HBV siRNA ). After poly( dA-dT ) stimulation, the expression of IFIT1 in the both cell lines were detected by real time RT-PCR. Results: Poly( dA-dT) was able to induce type I interferon production in HepG2 cells. The expression of IFN-β and IFIT1 peaked at 6 h and decreased gradually. TNF-α expression had no significant difference in compare with non-treatment group. TBK1 phosphorylation and IRF3 translocation could be detected, but no significant NF-KB activation was observed. In HepG2. 2. 15, the expression of IFN-β and IFIT1 increased gradually and peaked at 72 h, but TNF-α expression was transient at 12 h after poly( dA-dT) transfection. There were no translocation of NF-kB or IRF3 detected during 6h after treatment. HBV expression was inhibited after poly( dA-dT) and HBV replicative plasmid HBV1.3 co-transfection into HepG2 cells. However, HBV knockdown in HepG2. 2. 15 cells could up-regulate IFIT1 expression in response to poly( dA-dT) stimulation. Conclusion; Double-stranded DNA is able to elute type I interferon or pro inflammatory factor production in HepG2 and HepG2.2. 15 and the activation of NF-KB and IRF3 are involved in this signaling pathway. HBV can interfere with the innate immune response induced by dsDNA in hepatocyte-derived cells.%目的:了解乙型肝炎病毒(HBV)是否影响肝细胞对双链DNA(dsDNA)分子诱导产生的Ⅰ型干扰素应答.方法:以肝癌细胞HepG2以及整合有HBV基因的HepG2.2.15细胞为模型,转染dsDNA分子poly(dA-dT),实时定量RT-PCR检测IFN-β、干扰素应答基因IFIT1与炎症因子TNF-α的表达,Western blot检测NF-κB、IRF3的核转位以及pTBK1的表达.HBV复制型质粒HBV1.3转染HepG2、HBV siRNA转染HepG2.2.15后,实时定量RT-PCR检测细胞中IFIT1的表达.结果:poly(dA-dT)能够诱导HepG2细胞产生Ⅰ型干扰素,在转染后6小时,IFN-β与IFIT1表达达高峰,而后下降,TNF-α的表达与对照组相比没有明显变化.Poly(dA-dT)转染后6小时能检测到TBK1的磷酸化与IRF3的核转位,但NF-κB的核转位不明显.HepG2.2.15细胞在poly(dA-dT)转染后12小时能检测到TNF-α的一过性表达,而IFN-β与IFIT1的表达在72小时才达到高峰,与之对应的:在转染后6小时,没有检测到TBK1的磷酸化与IRF3的核转位.poly(dA-dT)与HBV1.3共转HepG2后,HBV的蛋白表达受到抑制,而将HBV siRNA转染HepG2.2.15后,细胞对poly(dA-dT)的应答明显增强.结论:HepG2与HepG2.2.15能够应对ds-DNA的刺激,通过NF-κB与IRF3信号通路产生Ⅰ型干扰素或炎症因子,但HBV的存在影响了肝细胞对dsDNA产生的固有免疫应答.