siRNA联合恩替卡韦在原代肝细胞中抑制土拨鼠肝炎病毒基因表达和复制  

Inhibition of Woodchuck Hepatitis Virus Gene Expression and Replication in Primary Hepatocytes by Combination of siRNA and Entecavir

作　　者：孟忠吉[1] 张永红[2] 柯昌征[1] 李东[1] 汤守兵[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1期35-38,43,共5页Journal of Hubei University of Medicine

基　　金：湖北省自然科学基金资助项目(2010CDZ036);湖北医药学院优秀中青年科技创新团队项目(2011CXX02)

摘　　要：目的:在原代土拨鼠肝细胞中研究siRNA联合恩替卡韦(ETV)对土拨鼠肝炎病毒(WHV)基因表达和复制的抑制作用。方法:分别设计合成针对WHV PreS1、S、C、X基因的4种siRNA,转染原代土拨鼠肝细胞,同时给予ETV处理,Southern和Northern印迹检测WHV病毒复制中间体和mRNA水平,realtime PCR检测细胞上清中WHV DNA。结果:4种siRNA对WHV基因表达和复制的作用显示出不同的动力学特征,siWHs和siWHx的抑制作用最强,而siWHpreS1和siWHc的活性相对较弱。在原代肝细胞中,首先出现的是WHV mRNA水平的降低,然后是WHV复制中间体水平的降低,siWHs和siWHx最高抑制60%～70%的mRNA和病毒复制中间体,同时抑制细胞上清中90%的WHV DNA。另外siWHx与ETV联合应用不仅可以增强抗病毒作用,而且可以防止ETV停药反跳。结论:siRNA在WHV自然感染的原代肝细胞中可以有效抑制病毒的基因表达和复制,因而siRNA有可能开发成为新的抗HBV药物,尤其适用于核苷(酸)类似物的联合应用。Objective To investigate the inhibition on woodchuck hepatitis virus(WHV) by combination of siRNA and Entecavir(ETV) in primary woodchuck hepatocytes(PWHs) with WHV natural infection.Methods Four siRNAs targeting the WHV preS1,S,C,and X region were synthesized and transfected into PWHs,in the presence or absence of ETV.The WHV gene expression and replication in PWHs and in the culture supernatants were determined by Northern blotting,Southern blotting,and realtime PCR,respectively.Results The four siRNAs led to a depletion of WHV transcripts and replicative intermediates with different kinetics.siWHs and siWHx were much more effective than siWHpreS1 and siWHc.In PWHs,WHV mRNAs were knocked down followed by WHV replicative intermediates.siWHs and siWHx had the highest efficacy to deplete 60%~70% of WHV transcripts and replicative intermediates,and about 90% of WHV DNA in the supernatants of PWHs.On the other hand,combination of siWHx with ETV exhibited more potently inhibition on WHV gene expression and replication,and prevented the rebound of WHV replication after the discontinuation of ETV.Conclusion siRNAs potently inhibited WHV gene expression and replication in PWHs with WHV natural infection,which might be useful as novel antiviral agents for the treatment of chronic HBV infection,especially when combined with nucleotide analogues.

关 键 词：RNA干扰 小干扰性RNA 土拨鼠肝炎病毒 原代肝细胞 核苷(酸)类似物 

分 类 号：R373.21[医药卫生—病原生物学]