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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。
Objective To investigate the mechanism of polyI (C), a ligand of Toll-like receptor 3 (TLR3), in inhibiting Hepatitis B virus (HBV) replication in human trophoblast cells. Methods Bewo cells were transfected with 2μg 1.3x HBV whole genome recombinant plasmid pcDNA3.1 (+) - HBV1.3, and treated with TLR3 ligand polyI: C for 3d after 12h. Bewo cells were then treated with polyI: C to observe the kinetics of IFN-β and TNF-α expression. Finally, the effect of nuclear factor-κB (NF-κB) antagonist PDTC on poly I: C-induced cytokine production in Bewo cells was observed. The levels of HBsAg, HBeAg and HBV DNA were detected by MEIA and fluorescence quantitative PCR respectively. The levels of IFN-β, TNF-α and the TIR domain adapter protein TRIF), myeloid differentiation protein 88 (MyD88) expression. Results Compared with the control group, poly I: C significantly inhibited HBV replication in Bewo cells (P <0.01), and poly I: C significantly induced the production of IFN-β and TNF-α in Bewo cells (P <0.05 ), In a time and dose-dependent manner. PDTC inhibited polyI: C-induced TNF-α production in cells, which was significantly lower than that in control group (P <0.01), but had no significant effect on IFN-β (P> 0.05). Compared with the control group, polyI: C induced TRIF mRNA expression in Bewo cells transfected with HBV recombinant plasmid (P <0.01). Conclusion Polyl: C TLR3 ligand may induce IFN-β and TNF-α production in Bewo cells via TRIF-dependent pathway. TNF-α production also involves NF-κB pathway and eventually inhibits HBV replication in Bewo cells.