靶向Nrf2基因RNA干扰真核表达载体的构建

目的：利用pSUPER载体构建针对人核因子E2 p45相关因子2（Nrf2）基因的RNA干扰真核表达载体，为研究Nrf2基因在结肠癌化学预防中的作用奠定实验基础。方法：设计特异性针对Nrf2基因的寡核苷酸序列及相应的对照序列，构建重组载体pSUPER Nrf2转染人结肠癌HT 29细胞。同时转染pEGFP N1质粒，通过荧光显微镜观察及流式细胞仪检测绿色荧光监测转染效率，共转染pEGFP N1 48h后G418筛选稳定表达的细胞。RT PCR和Western blot检测瞬时及稳定转染细胞Nrf2基因的表达，观察稳定筛选出的细胞中UGT1A基因mRNA水平的表达变化。结果：成功构建RNA干扰真核表达载体pSUPER Nrf2。转染后24～96?h，荧光显微镜及FCM检测显示转染效率为30％～75％。瞬时转染pSUPER Nrf2 A2，pSUPER Nrf2 B2重组质粒Nrf2 mRNA的表达差异无显著性（P>0.05）；瞬时转染72?h及稳定转染后，pSUPER Nrf2 A1，pSUPER Nrf2 B1可显著抑制Nrf2基因的表达。RT PCR检测显示，稳定筛选出的细胞中UGT1A表达水平降低（P<0.05）。结论：成功构建了Nrf2的RNA干扰表达载体，筛选并获得低表达Nrf2基因的稳定克隆，筛选出的细胞UGT1A表达水平明显降低，表明Nrf2可能对UGT1A酶的表达具有调节作用。

Construction of RNA interfering expression vector directed against Nrf2

Objective: To construct a RNAi expression vector aimed at human Nuclear factor E2 p45 related factor 2 (Nrf2) gene and it to study the chemoprevention for colon cancer. Methods：Two sequences targeting the ORF of Nrf2 were cloned into the RNA polymerase III based expression vector pSUPER. These recombinants were transfected into HT 29 cells. Fluorescence microscope and flow cytometry were used to determine the lipfectin transfection efficiency after being transfected with pEGFP N1 plasmids. The stable cells were selected in medium 48hours after pEGFP N1co transfected with G418. The expression of Nrf2 was assayed using RT PCR and Western blotting. RT PCR analysis of UGT1A mRNA was performed on the stable cells. Results：The construction of the recombinant expression vector pSUPER Nrf2 A1，B1 and its control vector pSUPER Nrf2 A2，B2 was successfully confirmed by the results of enzyme digestion, electrophoresis and sequencing. The transfection efficiency was 30％～75%. The ability of these vectors inhibiting Nrf2 in a transient and stable expression experiment in HT 29 cells was compared.Importantly, pSUPER Nrf2 B1 was able to significantly knockdown Nrf2 expression. pSUPER Nrf2 A1 only had a moderate activity, whereas pSUPER Nrf2 A2，B2 were inactive in this assay. Moreover, activities of UGT1A was reduced by 20％～30％ in the stable cells transfected with pSUPER Nrf2 A1，B1 vector. Conclusion：siRNA expression mediated by the pSUPER vector causes efficient, stable, and specific down regulation of Nrf2 gene expression，suggesting the suppression of Nrf2 gene expression results in down regulation of the constructive expression of UGT1A gene.