摘要
目的探讨维甲酸诱导基因-I(RIG-I)在诱导HBV感染IFN产生中的影响,以期对阐明乙型肝炎慢性化机制,寻找新的治疗靶点提供新的思路。方法以RIG-I表达载体flagRIG-l-ful1转染培养6孔板中的HepG2、HepG2.2.15,24h后用水疱性口炎病毒(VSV)感染细胞,然后在0、8、16和24h收集细胞及培养上清液,采用quantitive RT-PCR、Western印迹检测RIG-I、MDA5、IPS-1、ISG54等基因的表达,ELISA检测培养上清液中分泌的IFN-β水平。结果 HepG2.2.15细胞在VSV感染后IFN-β分泌水平(11.18±1.34)pg/mL明显低于HepG2细胞(275.50±22.97)pg/mL(P<0.01);通过质粒flagRIG-I-ful1转染高表达RIG-I后,HepG2.2.15分泌IFN-β的能力恢复至(548.78±57.99)pg/mL与HepG2细胞(532.10±39.34)pg/mL接近的水平(P=0.7013)。结论 HepG2.2.15细胞感染VSV后IFN产生障碍,高表达RIG-I后IFN表达水平得到恢复,提示RIG-I功能缺陷导致抗病毒免疫应答降低,RIG-I可能在HBV感染清除中起重要作用。
Objective The retinoic acid-inducible gene I product (RIG-I) has been identified as a cellular sensor of virus infection resulting in beta interferon (IFN-β) induction. Our previous study showed that hepatitis B virus (HBV) infection could disturbed RIG-I expression in moDC from CHB patients. This study aims to investigate the function of RIG-I in inducing 1FN production with HBV infection, and to help understand the mechanism of chronic hepatitis B infection. Methods HepG2 and HepG2. 2. 15 cells were transfected with RIG 1 plasmid flag RIG I-full using Lipofectamine 2000 (Invitrogen). After 24 h over-expression of RIG-I, HepG2.2. 15 and HepG2 cells were also infected with VSV at MOI of 0. 1. At 0, 8, 16 and 24 h, RIG-I, 1PS-1 and IFN-β levels in these cells were detected by quantitative Real time PCR, western blot and EI.ISA respectively. Results HepG2. 2. 15 (11. 18 ± 1. 34) pg/mL produced much less 1FN-~ than HepG2 (275. 50 ± 22. 97) pg/mL 24 h after flagRIO-l-full transfectlon, cells and cultured supernatants were harvested and analyzed. It was found that in RIO-I overexpressed cells, IFN-β in transfected HepG2.2.15 (548.78 ± 57. 99) pg/mL was similar with that in transfected HepG2 cells (532. 10 ± 39.34) pg/mL (P= 0. 7013). Conclusion RIG-I could restore IFN-β secretion suppressed by HBV in HepG2. 2. 15 cells, which indicated confirm that RIG-I played an important role in the clearance of chronic HBV infection.
出处
《肝脏》
2012年第8期554-557,共4页
Chinese Hepatology
基金
国家自然科学基金资助项目(30671838
30872252
81070334)
十一五"艾滋病和病毒性肝炎等重大传染病防治"科技重大专项课题资助项目(2008ZX10002-005
2008ZX10002-007)