Nrf2过表达与敲减Hep1-6稳转细胞株的建立

摘要

Nuclear factor NF-E2 related factor 2 (Nrf2) is an important target molecule in nonalcoholic fatty liver dis-ease ( NAFLD) . To study the role of small molecules that targets Nrf2 in vitro, mice Nrf2 gene cloned by PCR was cloned in-to pLenti6/V5-D-TOPO®empty carrier,building a pLenti6/V5-Nrf2 reorganization plasmid which was identified via sequen-cing and restriction enzyme digestion. The plasmid was packed into lentivirus and used to infect Hep1-6 liver cancer cells. Meanwhile, the shRNA-Nrf2 plasmid was packed into lentivirus and used to infect Hep1-6 liver cancer cells as well. The cells were screened with blasticidin or puromycin for about 1 month. The qPCR results showed that the expression of Nrf2 was four times than that of control in Nrf2 over-expression cells while it was significantly reduced in Nrf2 knock down cells. Con-sistently, the level of protein in the two cell lines detected by Western blotting was obviously increased in Nrf2 over-expression cells or reduced in Nrf2 knock down cells. The results suggest that the Nrf2 over expression and knock down Hep1-6 cell lines have been successfully built and it supplies a useful tool to study the role of drug targeting Nrf2 in vitro.%核因子NF-E2相关因子2(Nrf2)是治疗非酒精性脂肪肝(NAFLD)的一个重要靶点分子。为了从体外研究小分子通过靶向Nrf2改善NAFLD的分子机制,首先PCR克隆鼠源Nrf2基因至pLenti6/V5-D-TOPO襅空载体得到重组载体pLenti6/V5-Nrf2,通过测序、酶切鉴定正确后,扩繁质粒,制备慢病毒并将其感染Hep1-6肝癌细胞,用稻瘟醇胚素筛选、鉴定获得稳定表达Nrf2的细胞株。同时,用Nrf2干扰质粒构建慢病毒感染Hep1-6肝癌细胞,通过嘌呤霉素筛选、鉴定Nrf2敲减的细胞株。 Q-PCR检测结果显示,过表达稳转株( pLenti6/V5-Nrf2)中Nrf2基因的表达量为对照组( pLenti6/V5-LacZ)的4倍,敲减稳转株中Nrf2基因的表达量大幅度降低。 Western blotting显示过表达稳转株的Nrf2表达量明显高于对照组,敲减稳转株的Nrf2表达量明显低于对照稳转株,此结果与Q-PCR结果一致。所克隆的Nrf2基因和敲减基因在Hep1-6小鼠肝癌细胞中得到了正确转录和翻译,稳转细胞株构建成功。