【摘要】 目的:构建慢病毒介导的人孕烷X受体(hPXR)sh-RNA干扰载体及筛选肝癌细胞稳定干扰株。方法:针对hPXR的特异性序列,设计干扰序列,构建重组克隆。转染HEK293T细胞,筛选有效干扰片段。HEK293T包装干扰病毒并转染肝癌HepG2细胞株,200 mmol/L嘌呤霉素筛选2-3周,Western blot检测PXR表达。hPXR激动剂利福平处理PXR sh-RNA稳定干扰肝癌HepG2细胞株和Scramble对照细胞,抽提RNA并鉴定hPXR靶基因细胞色素P450 3A4(CYP3A4)的表达。结果:筛选出了PXR有效干扰片段,并获得了PXR信号通路有效敲低的HepG2慢病毒稳定干扰株。结论:成功构建了慢病毒介导的PXR sh-RNA稳定干扰HepG2细胞株。更多还原

【Abstract】 OBJECTIVE: To establish a HepG2 cell line with stable PXR silencing effect by a lentiviral vector carrying a short hairpin RNA(sh-RNA). METHODS:Three double-stranded sh-RNA targeting the PXR gene were designed,synthesized and cloned into the psi-sH1 sh-RNA vector. The effective recombinant sh-RNA expression plasmid against PXR,which was selected of HEK293T cells with three double-stranded sh-RNA plasmids,was packaged into HEK293T cells and used to infect HepG2 cells after 200 mmol/L puromycin screening for 2-3 weeks. The expression level of PXR was detected by western blot,the CYP3A4 mRNA levels both in sh-RNA PXR HepG2 stable cells and scramble control HepG2 cells were detected by real-time PCR. RESULTS:A recombinant sh-RNA expression plasmid against PXR gene with effective interference was successfully selected and a HepG2 cell line stably transfected with the sh-RNA PXR was established. CONCLUSION:A HepG2 cell line stably transfected with the sh-RNA P XR was established.更多还原