TLR3配体polyI:C经TRIF/NF-κB途径抑制Bewo细胞中HBV复制

TLR3 ligand polyI:C-mediated inhibition hepatitis B virus replication in Bewo cells via TRIF/NF-κB pathway

目的探讨Toll样受体3(TLR3)配体聚肌苷酸胞苷酸(polyI:C)抑制人滋养层细胞Bewo中乙型肝炎病毒(HBV)复制的作用机制。方法首先将2μg 1.3倍HBV全基因重组质粒pcDNA3.1(+)-HBV1.3转染Bewo细胞,12h后,以TLR3配体polyI:C处理3d。然后以polyI:C处理Bewo细胞,观察IFN-β、TNF-α表达的动力学。最后,观察核因子-κB(NF-κB)拮抗剂PDTC对polyI:C诱导Bewo细胞产生细胞因子的作用。采用微粒子酶免疫分析法(MEIA)和荧光定量PCR法分别检测HBsAg、HBeAg和HBV DNA水平,并以ELISA和RT-PCR分别检测IFN-β、TNF-α水平及TIR结构域的转接蛋白(TRIF)、髓样分化蛋白88(MyD88)表达。结果与对照组比较,polyI:C可显著抑制Bewo细胞中HBV复制,差异有统计学意义(P<0.01),且polyI:C可显著诱导Bewo细胞产生IFN-β和TNF-α(P<0.05),呈时间和剂量依赖性。PDTC可抑制polyI:C诱导细胞产生TNF-α,显著低于对照组(P<0.01),但对IFN-β无显著作用(P>0.05)。与对照组比较,polyI:C可诱导HBV重组质粒转染的Bewo细胞表达TRIF mRNA(P<0.01)。结论 TLR3配体polyI:C可能通过TRIF依赖性途径诱导Bewo细胞产生IFN-β和TNF-α,且TNF-α产生还涉及NF-κB途径,并最终抑制Bewo细胞中HBV复制。

Abstract：Objective To explore the effect and mechanism of Toll-like receptor 3 （TLR3） ligand polyinosinie polycytidylic acid （polyI：C）-mediated inhibition hepatitis B virus （HBV） replication in Bewo cells. MethOds Firstly, 2 p. g 1.3-fold HBV recombinant vector pcDNA3.1 （＋）-HBV1.3 were transfected into Bewo cells,after 12h, the cells were treated with polyI：C for 3d. Then Bewo ceils were exposed to TLR3 ligand polyI：C to observe the kinetics of IFNand TNF-ctexpression in Bewo cells.Eventually,to observe the effect of pyrrolidine dithioearbamate （PDTC）,an inhibitor of NF-KB,on polyI：C-induced eytokines. To detect HBsAg,HBeAg and HBV DNA level in the culture supematant by Microparticle Enzyme Immunoassay （MEIA） and fluorescence quantitative PCR,respeetively,and to assay IFN-β TNF-otlevel and ＇adaptor protein TRIF,myeloid differential factor 88 （MyD88） expression by ELISA and RT-PCR, respectively. Results Compared with control,polyI：C could significantly suppresse HBV replication （P〈0.01）,and it could striking induce the production of IFN-β and TNF-a in Bewo cells（P〈0.05 ）,in time- and dose-dependent manners. Compareds with control,PDTC strongly inhibited polyI：C to induce TNF-a preduction,not IFN-β in Bewo cells（P〈0. 01）.The mRNA levels of TRIF were significantly induced by polyI：C in the Bewo cells transfected with this recombinant vector（P〈0.01 ）.Conclusions These findings suggest that TLR3 ligand polyI：C could significantly suppress HBV replication by inducing IFN-β and TNF-a production in Bewo cells via the TRIF-dependent pathway,and secretion of TNF-a still involves the NF- K B pathway.