摘要
目的探讨肝细胞的Toll样受体（TLR）信号途径及其诱导的抗病毒免疫应答。方法分离野生型C57BL／6小鼠的原代肝细胞，定量逆转录一聚合酶链反应法检测TLR的表达。分别用TLR1～9配体刺激肝细胞并收集细胞上清液。酶联免疫吸附法检测细胞上清液内的细胞因子。病毒保护实验检测细胞上清液的抗脑膜炎心肌炎病毒因子，并将细胞上清液与HBV-Met细胞共孵育，用Southernblot法检测其对HBV复制的抑制效应。结果原代肝细胞能表达TLR1～9。与其TLR表达谱相应的，肝细胞在TLR1～9配体的刺激下均可以产生炎性细胞因子（肿瘤坏死因子α和白细胞介素6），而仅在TLRl、TLR3、TLR7和TLR9配体刺激下可产生I型干扰素（干扰素α和干扰素β）。在病毒保护实验中，TLR3和TLR7的配体可以刺激肝细胞产生大量的抗脑膜炎心肌炎病毒效应分子；而TLRl、TLR3和TLR4配体直接刺激的肝细胞上清液，以及TLR3、TLR7和TLR9配体转染刺激的肝细胞上清液，都能有效抑制HBV的复制。结论小鼠原代肝细胞有独特的TLR信号途径，并能通过TLR配体的激活产生抑制HBV复制的效应。这一发现对于制定基于TLR的抗肝脏靶向性病毒的治疗措施有指导意义。
Objective This report aims to investigate the Toll-like receptor （TLR） signaling pathways and induced antiviral activity in hepatocytes. Methods We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supematants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay （in EMCV system） and the control of HBV replication were assessed via Southern blotting （in HBV system）. Results We demonstrated that -hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines （ TNF-α or IL-6 ）, to TLR -1,-3,-7 and -9 ligands by producing type I IFN （ IFN-α or IFN-β ）. Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, superuatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication. Conclusion primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropism virus.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2011年第11期838-842,共5页
Chinese Journal of Hepatology
基金
国家传染病防治重大专项（2008ZXl0002-011）
国家重点基础研究发展计划（“973”计划）项目（2007CB512804,2009CB522500）
科技部国际合作与交流专项（2011DFA31030）
国家自然科学基金（81001313）
中国博士后科学基金（20090460949）
