目的 研究持续表达的丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)对HepG2细胞内质网应激反应的影响.方法 NS4B重组真核表达质粒pcDNA3.1(-)NS4B通过脂质体转染HepG2细胞,G418筛选、基因组PCR和Western blot鉴定稳定转染细胞;RT-PCR检测稳定转染细胞内人X盒结合蛋白1(XBP1) mRNA剪接,荧光素酶试验检测稳定转染细胞内GRP78启动子、XBP1启动子和核因子κB(NF-κB)活性.结果 基因组PCR和Western blot鉴定证实NS4B基因整合到转染HepG2细胞的染色体上,并有效表达;在NS4B稳定转染的HepG2细胞中,检测到XBP1 mRNA剪接、XBP1和Grp78启动子激活、Ca2+稳态变化及NF-κB激活.结论 NS4B在HepG2的稳定表达诱导了内质网应激反应,揭示NS4B可能通过诱导肝细胞内质网应激反应在HCV感染及其致病性中发挥作用.%Objective To study the regulation of endoplasmic reticulum(ER) stress response by continuous expression of NS4B in HepG2 cells. Methods The recombinant vector pcDNA3. 1( - ) NS4B carrying hepatitis C virus NS4B gene was transfected to HepG2 cells using Lipofectamine 2000 reagent kit. Stable HepG2 cell lines with an integrated NS4B gene were selected by G418 and then confirmed by genome PCR and Western blot analysis;XBPl mRNA splicing,GRP78 and XBP1 promoter activation,Ca2+ ho-meostasis change and NF-kB activation were analyzed in stably-transfected HepG2 cells using RT-PCR and luciferase assays. Results Genome PCR and Western blot demonstrated together that NS5B gene was integrated into the chromosome of transfected HepG2 cells and expressed successfully;XBP1 mRNA splicing,GRP78 and XBP1 promoter activation,Ca2+ homeostasis change and NF-κB activation were observed in HepG2 cells stably-transfected with pcDNA3. 1 ( - ) NS4B. Conclusion Continuous expression of NS4B in HepG2 cells induces ER stress response, which implies the role of NS4B in HCV infection and HCV-associated liver disease via the induction on ER stress response.
展开▼
