靶向Nrf2基因shRNA慢病毒载体的构建及其对肝星状细胞Nrf2基因表达的沉默

Construction of short hairpin RNA lentiviral vector targeting Nrf2 gene and its silencing effect on Nrf2 gene in HSC-T6 cells

目的设计以NF-E2-related factor2(Nrf2)基因为靶点的短发夹状RNA(shRNA),构建重组慢病毒表达载体并转染大鼠肝星状细胞株HSC-T6,观察其对Nrf2表达的影响。方法应用重组DNA技术,将设计好的4条基因特异性shRNA序列插至慢病毒表达载体p GLV3-GFP中,构建p GLV3-GFP-Nrf2-shRNA-1/2/3/4,并应用脂质体法转染293T细胞,进行病毒包装及滴度测定。采用实时荧光定量PCR(RT-PCR)及Western blot分别从mRNA和蛋白水平检测转染72 h和96 h后大鼠肝星状细胞株HSC-T6 Nrf2基因的表达情况。结果测序证实慢病毒载体构建成功,并测定滴度为1×109TU/ml,p GLV3-GFP-Nrf2-shRNA转染后72 h和96 h可抑制Nrf2基因mRNA及蛋白的表达。结论成功构建Nrf2基因的shRNA慢病毒载体,可有效抑制大鼠肝星状细胞株HSC-T6中Nrf2基因的表达。

Objective To construct lentiviral vector targeting NF-E2-related factor2 （Nrf2）gene and evaluate its silencing effect on Nrf2 gene in rat hepatic stellate cell line HSC-T6.Methods Four short hairpin RNA （shRNA）fragments targeting Nrf2 were designed and cloned into lentiviral vector pGLV3-GFP to construct pGLV3-GFP-Nrf2-shRNA1/2/3/4.Then the silencing effects on Nrf2 gene were confirmed by real-time PCR and Western blot in transfected HSC-T6 cells at 72 h and 96 h.Results The Nrf2 shRNA lentiviral vectors were successfully constructed and confirmed by sequencing when the virus reached a titer of 1 × 109 TU/ml.The expression of Nrf2 in both mRNA and protein levels were inhibited by real-time PCR and Western blot.Conclusion The shRNA expressing lentiviral recombinants targeting the Nrf2 gene are successfully constructed and could effectively silence expression of Nrf2 gene in HSC-T6 cells.