乙型肝炎病毒核心抗原通过Toll样受体4促进HepG2．2．15细胞的侵袭

Hepatitis B core antigen promotes invasion of hepatocellular carcinoma cell line HepG2.2.15 via Toll-like receptor 4

目的探讨乙型肝炎病毒核心抗原（HBcAg）促进HBV相关肝癌细胞系HepG2．2．15细胞侵袭的效应及Toll样受体4（TLR4）在其中发挥的作用。方法应用反转录实时定量PCR法和Westernblot法检测HepG2细胞和HepG2．2．15细胞中TLR4的表达水平。TLR4特异性小干扰RNA（siRNA）转染HepG2．2．15细胞以沉默TLR4，并用重组HBcAg刺激培养，利用Transwell嵌入式平板培养法观察细胞侵袭，并检测培养细胞中TLR4信号通路相关蛋白的表达和培养上清液中促炎细胞因子的表达情况。据资料不同分别采用Studentf、One-WayANOVA和SNK-q检验进行统计学分析。结果HepG2．2．15细胞中TLR4mRNA和蛋白的表达水平均较HepG2细胞中明显升高。TLR4siRNA转染可显著降低HepG2．2．15细胞中TLR4的表达水平。抑制TLR4表达和（或）HBcAg刺激均不影响HepG2．2．15细胞增殖。但是，HBcAg刺激可显著提高HepG2．2．15细胞侵袭能力和促炎细胞因子（包括干扰素Y，白细胞介素1D、6、8和肿瘤坏死因子α）的分泌，HBcAg刺激后的侵袭细胞数[（386±36）个]较未刺激组[（98±10）个]明显增加（P=0．0002）；而抑制TLR4表达可显著降低HBcAg介导的细胞侵袭[（205±16）个对比（386±36）个]，P=0．0013。同时，HBcAg刺激可增加HepG2．2．15细胞中MyD88和TLR衔接蛋白的表达，而TLR4基因沉默可抑制HBcAg诱导的MyD88的表达升高，但对TRIF的表达无明显影响。结论HBcAg可促进HepG2．2．15细胞的侵袭，TLR4／MyD88信号通路可能通过诱导促炎细胞因子的表达参与这一过程。

Objective To investigate the effect of hepatitis B core antigen （HBcAg） in promoting the invasion of hepatitis B virus （HBV）-related hepatocellular carcinoma cell line HepG2.2.15 and the role of Toll-like receptor 4 （TLR4） in the mechanism. Methods TLR4 mRNA and protein expression in HepG2 cells and HepG2.2.15 cells was measured by reverse transcription real-time PCR and Western blot analysis, respectively. HepG2.2.15 cells were transfected with TLR4 specific small interfering RNA （siRNA） to silence TLR4 expression, and stimulated by recombinant HBcAg in culture. The invasion of cells was measured by Transwell invasion assay. The expression of TLR4 signaling pathway-related proteins in the cultured cells and proinflammatory cytokines in the supernatant was also determined. The student＇s t-test, one-way ANOVA, and SNK-q test were used for statistical analysis. Results TLR4 mRNA and protein expression in HepG2.2.15 cells was significantly higher than that in HepG2 cells. TLR4 siRNA transfection remarkably down-regulated TLR4 expression in HepG2.2.15 cells. Inhibiting TLR4 expression and/or HBcAg stimulation did not affect the proliferation of HepG2.2.15 cells. However, HBcAg stimulation significantly increased the invasion ability of HepG2.2.15 cells and the secretion of proinflammatory cytokines [including interferon （IFN）-γ, interleukin （IL）-6, IL-8, and tumor necrosis factor （TNF）-α]. Inhibiting TLR4 expression significantly reduced HBcAg-induced cellular invasion. Meanwhile, HBcAg stimulation elevated the expression of MyD88 and TRIF in HepG2.2.15 cells. TLR4 silencing inhibited HBcAg-induced increase in the expression of MyD88, while it showed no significant impact on TRIF expression. Conclusion HBcAg can promote the invasion of HepG2.2.15 cells. The TLR4/MyD88 signaling pathway may be involved in this process by inducing the expression ofproinflammatory cytokines.