RNAi通过PKR和TLR通路活化天然免疫  

RNAi Enhances Innate Immunity Through PKR and TLR Signalling Pathways

作　　者：孟忠吉[1] 张永红[2] 李东[1] 汤守兵[1] 柯昌征[1] 陈悦[1] 

机构地区：[1]湖北医药学院附属太和医院感染科,湖北十堰442000 [2]湖北医药学院附属太和医院中西医结合科,湖北十堰442000

出　　处：《湖北医药学院学报》2013年第6期461-464,472,共5页Journal of Hubei University of Medicine

基　　金：湖北省自然科学基金(2010CDZ036);湖北医药学院优秀中青年科技创新团队项目(2011CXX02);湖北医药学院国家级科研项目培育基金(2012GPY10)

摘　　要：目的:研究特异性RNA干扰(RNA interference,RNAi)上调天然免疫的分子机制。方法:分别从土拨鼠肝炎病毒(Woodchuck hepatitis virus,WHV)转基因小鼠和WHV慢性感染的土拨鼠分离原代肝细胞,转染针对WHV和小鼠甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的小干扰RNA(small interfering RNA,siRNA),同时用特异性siRNA抑制剂和信号通路抑制剂,研究RNA活化蛋白激酶(RNA-activated protein kinase,PKR)、Toll样受体(toll-like receptor,TLR)3、7/8和视黄酸诱导基因Ⅰ(Retinoic acid-inducible gene-Ⅰ,RIG-Ⅰ)/黑色素瘤分化相关基因5(Melanoma differentiation-associated gene-5,MDA5)等信号通路在RNAi上调天然免疫中的作用,Real-time RT PCR检测靶基因和MxA的mRNA水平,Western blot检测信号通路分子磷酸化PKR、核因子κB(Nuclear FactorκB,NF-κB)和干扰素调节因子(interferon regulator factor,IRF)3的蛋白水平。结果:针对WHV和GAPDH的RNAi介导的黏病毒抵抗蛋白A(myxovirus resistance A,MxA)上调可以被PKR抑制剂和内体酸化抑制剂所阻断,特异性RNAi可以诱导PKR磷酸化和NF-κB与IRF3的核内转位。结论:特异性RNAi介导的靶RNA降解产物可能通过PKR和TLR-3,-7/8通路活化天然免疫。Objective To investigate the mechanism by which RNAi enhances innate immunity. Methods Primary murine hepatocytes( PMHs) from WHV transgenic( Tg) mice and primary woodchuck hepatocytes( PWHs) from woodchucks with WHV chronic infection were prepared and treated with siRNAs targeting WHV or mouse GAPDH,in the presence or absence of inhibitors of PKR( 2-AP and PKR-I) and TLR-3,-7 /8( chloroquine) pathways,or siRNAs targeting RIG-I / MDA5. The mRNA levels of target genes and MxA were detected by real-time RT-PCR. The protein levels of phosphorylated PKR, NF-κB p65 subunit and IRF3 were determined by Western blotting. Results The RNAi mediated MxA upregulation could be abolished by the inhibitors of PKR and TLR-mediated signaling pathways,but not by the interference of RIG-I / MDA5 signaling pathway. WHV specific RNAi resulted in an increase in PKR phosphorylation and nuclear translocation of IRF3 and NF-κB. Conclusion RNAi directed small RNA cleavage products may enhance innate immune responses through the PKRand TLR-dependent signaling pathways.

关 键 词：RNA干扰 土拨鼠肝炎病毒 原代肝细胞 天然免疫 

分 类 号：R392.1[医药卫生—免疫学]