TLR9受体介导枯否细胞对转染HepG2细胞HBx信号转导的抑制作用  

Inhibitory effect of Kupffer cells on HBx-mediated signaling in HepG2 cells via Toll-like receptors-9

作　　者：范熠[1] 唐宇[1] 高燕[1] 

机构地区：[1]第三军医大学附属西南医院全军感染病研究所,重庆市400038

出　　处：《实用肝脏病杂志》2010年第1期9-12,共4页Journal of Practical Hepatology

摘　　要：目的探讨胞嘧啶鸟嘌呤二核苷酸-脱氧寡核苷酸激活类铎受体9信号通路抑制肝脏肿瘤细胞增殖的免疫机制。方法首先将乙型肝炎病毒X基因真核表达质粒转染入HepG2细胞,以空质粒转染HepG2细胞为对照组,24h后以β-actin为内参照对HBx蛋白表达进行Westernblotting鉴定;然后分别将CpG或CpG对照和枯否细胞加入转染HepG2细胞培养体系中进行共培养,12h后收集上清液,采用ELISA法检测干扰素(IFN)-α和IFN-γ,同时收集贴壁的HepG2细胞进行双荧光素酶活性检测。结果Westernblotting鉴定显示转染的HepG2细胞表达特异性HBx蛋白;双荧光素酶活性检测显示KC细胞与CpG加入转染HepG2细胞培养体系后HBx信号转导活性下降35%;CpG激活的KC细胞产生大量的IFN-α和IFN-γ,与对照组相比分别上升了8.7倍和6.5倍。结论CpG激活KC细胞产生多种干扰素,抑制HBx信号转导,这为通过阻断HBx信号转导治疗肝癌提供了理论依据。Objective To investigate the inhibitory immune mechanisms of cytidine-phosphate-guanosine oligodeoxynucleotides（CpG） on liver cancer proliferation via Toll-like receptors-9（TLR9）. Methods The recombinant expression plasmid of hepatitis B virus X （HBx） were transfected into HepG2 cells and the blank plasmidtransfected HepG2 cells was the control group. 24h later,the HBx protein and the β-actin control protein were identified in the HepG2 cells by Western blotting. Then,CpG and Kupffer cells （KC）,CpG-control and KC were added into the transfected HepG2 cell culture system,respectively. 12h later,the snpernatant were collected to detect interferon （IFN）-α and IFN-γ by ELISA,and the adherent HepG2 cells were collected to detect luciferase by reporter gene assay system. Results The Western blotting results showed that the HBx plasmid-transfected HepG2 cells expressed the specific HBx protein; and the dual luciferase reporter gene assay showed that the activity of HBx-mediated signaling decreased by 35% when adding KC cells and CpG into the HBx plasmid-transfected HepG2 cell culture system;and CpG activated KC cells to produce IFN-α and IFN-γ , which were increased by 8.7 folds and by 6.5 folds respectively compared with the control group. Conclusion CpG-activated KC cells could inhibit HBx-mediated signaling through the production of IFN-α and IFN-γ. It might provide a theoretical basis for HCC therapy by blocking the HBx-mediated signal pathway.

关 键 词：HEPG2细胞 乙型肝炎病毒X蛋白 枯否细胞 类铎受体 

分 类 号：R73-3[医药卫生—肿瘤]