靶向Nrf2基因RNA干扰真核表达载体的构建  

Construction of RNA interfering expression vector directed against Nrf2

作　　者：杨晓云[1] 李延青[1] 梁晓红[2] 郭玉婷[1] 袁俊华[1] 张燕[1] 朱强[1] 

机构地区：[1]山东大学齐鲁医院消化内科,山东济南250012 [2]山东大学免疫学研究所,山东济南250012

出　　处：《山东大学学报（医学版）》2006年第4期345-350,共6页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助课题(30370634/C03030204)

摘　　要：目的：利用pSUPER载体构建针对人核因子E2p45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。方珐：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-N148h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pSU-PER-Nrf2。转染后24-96h，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72h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。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-Nlco-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 - 2 9 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.

关 键 词：基因 Nrf2 尿苷二磷酸葡萄糖醛酸转移酶 RNA干扰 结肠肿瘤 

分 类 号：Q786[生物学—分子生物学]