CRISPR/Cas9敲除HBV pre S1段对肝癌细胞中HBV病毒复制的影响  

Effect of HBV pre S1 domain knockout with CRISPR/Cas9 on HBV replication in hepatocellular carcinoma cells

作　　者：龙黎 罗新华[1] 张茜[1] LONG Li;LUO Xinhua;ZHANG Qian(Department of Infectious Diseases,Guizhou Provincial People's Hospital,Guiyang 550002,Guizhou,Ch)

机构地区：[1]贵州省人民医院感染科,贵州贵阳550002

出　　处：《贵州医科大学学报》2022年第10期1163-1168,1188,共7页Journal of Guizhou Medical University

基　　金：贵州省科技计划项目(黔科合基础-ZK[2021]重点013);贵阳省卫生健康委科学技术基金项目(gzwjkj2020-1-043)。

摘　　要：目的 探讨CRISPR/Cas9基因编辑技术敲除HBV pre S1段对肝癌细胞中HBV病毒复制的影响,分析基因编辑联合传统抗病毒药物或免疫调节剂的抗病毒疗效。方法 根据HBV pre S1段设计特异性干扰片段g RNA(sg RNA-1、sg RNA-2及sg RNA-3)接入表达载体进行转化,经DAPI染色鉴定转染、获得HBV pre S1敲除的Hep AD38细胞;采用马酸替诺福韦(TDF)和干扰素-α(IFN-α)处理HBV pre S1敲除的Hep AD38细胞,取上清液、采用酶联免疫吸附实验(ELISA)检测HBs Ag、HBcr Ag的含量,RT-PCR方法检测HBV DNA、pg RNA水平。结果 成功构建HBV g RNA,sg RNA-3作用最强;成功获得HBV pre S1敲除的Hep AD38细胞,Cas9/sg RNA定位于细胞核;与对照组细胞比较,HBV pre S1敲除的Hep AD38细胞HBV复制水平降低(P <0.05),TDF或IFN-α处理HBV pre S1敲除的Hep AD38细胞能有效增强抗病毒效应(P <0.05),且IFN-α处理的细胞更能抑制HBV RNA和HBs Ag的分泌。结论 Cas9/sg RNA靶向HBV DNA切割能抑制HBV病毒的复制,传统抗病毒药物或免疫调节剂能使该作用增强。Objective This paper sought to explore CRISPR/Cas9 genome editing technology specifically targeting the hepatitis B virus(HBV)pre S1 segment for gene silencing in a cellular model of HBV and further compare the antiviral efficacy of genome editing combined with traditional antiviral drugs or immunomodulators.Methods Changes in HBV and antigen secretion levels in an in vitro model of chronic HBV infection were observed using lentiviral transduction of a bacterial Cas9 gene and single guide RNAs(sgRNAs)specific for HBV.Results Firstly,HBV gRNA was successfully constructed.Next,Cas9/sgRNA was transduced into cells,validating that Cas9/sgRNA was localized in the nucleus.The cell supernatants were harvested to measure the levels of HBV markers.The comparison revealed that the level of HBV replication was significantly lower in the cells intervened by sgRNA compared with the control groups(P<0.05).Finally,the combination treatment of sgRNA-3 with tenofovir(TDF)or interferon-alpha(IFN-α)could effectively enhance the antiviral effects(P<0.05).Furthermore,sgRNA-3 combined with IFN-αwas superior to sgRNA-3 combined with TDF for suppressing the secretion of HBV RNA and HBsAg.Conclusions This paper demonstrates the repressive effects of Cas9/sgRNA on virus replication via HBV DNA cleavage using a model with chronic HBV infection.This new observation provides substantial support for the bacterial CRISPR/Cas system as a potential approach for the treatment of DNA virus infections.

关 键 词：乙型肝炎病毒 抗病毒治疗 CRISPER/Cas9 病毒复制 临床治愈 基因编辑 

分 类 号：R575.1[医药卫生—消化系统]