质粒介导的短发卡状RNA特异性抑制大肠癌cerb-B1基因的表达  

Inhibitory effects of short hairpin RNA mediated by plasmid vector on cerb-B1 gene expression in human colon cancer cells

作　　者：吴相柏 邬跃刚 宋文军 曹政 吴利达 陶凯雄[2] 

机构地区：[1]宜昌市第二人民医院外科,湖北宜昌443000 [2]华中科技大学同济医学院附属协和医院普通外科,湖北武汉430022

出　　处：《南京医科大学学报（自然科学版）》2007年第2期137-141,F0003,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：湖北省科技攻关计划资助项目(2005A304B09)

摘　　要：目的:体外构建编码人类基因cerb-B1短发卡状RNA(shorthairpinRNA,shRNA)的质粒表达载体,观察其对结肠癌LoVo细胞cerb-B1基因和表皮生长因子受体(EGFR)蛋白的特异性抑制作用。方法:体外合成cerb-B1基因的DNA模板引物,和Pgenesil-1质粒构建编码shRNA的表达载体。应用脂质体Lipofectamine2000转染人结肠癌LoVo细胞,转染成功后以G418筛选4周,实时荧光定量RT-PCR(RealTimeRT-PCR)检测cerb-B1基因的mRNA表达,Western-blot检测EGFR蛋白的表达。结果:质粒载体经酶切鉴定证实DNA模板插入质粒成功,且测序与设计结果相符,成功的构建了针对原癌基因cerb-B1的质粒表达载体。质粒载体成功转染后,cerb-B1基因mRNA表达下降了(81.3±2.8)%,EGFR蛋白表达下降了(73.4±2.3)%,和对照质粒载体组比较有显著性差异。结论:说明本研究构建的针对cerb-B1基因的质粒表达载体可以显著抑制cerb-B1基因和EGFR蛋白在人结肠癌LoVo细胞的表达,为结肠癌的基因治疗提供了新的思路。　 Objective：To establish a plasmid expressive vector coding short hairpin RNA（shRNA） of human cerb-B1 gene in vitro, and to investigate specific inhibition of cerb-B1 gene and epidermal growth factor receptor（EGFR） expression in human colon cancer LoVo cells by shRNA, nethods：DNA templa-primer of cerb-B1 gene was synthetized in vitro and the plasmid expression vectors coding shRNA were established with Pgenesil-I plasmid. The human colon cancer LoVo cells were transfected by Lipofectamine 2000 with plasmid vector, and then selected for 4 weeks by G418. The expression of cerb-B1 mRNA was assessed by real time PCR and the protein of EGFR was assessed by Western blot method. Results：The digest identification of plasmid vectors confirmed that DNA templa-primer was successfully inserted in the plasmid, and the sequence was in conformity with the designed result. Plasmid expression vectors aiming directly at cerb-B1 gene were successfully established. After the plasmid vectors were transfected into the LoVo cells, the expression of cerb-B1 mRNA was decreased by（81.3± 2.8）%, and the expression of EGFR protein was decreased by （73.4 ± 2.3）%, respectively. There was a significant difference to the control plasmid vectors. Conclusion：The plasmid expression vector aiming directly at cerb-B1 gene might suppress cerb-B1 gene and EGFR expression in human colon cancer LoVo cells. The new therapeutic modalities could be used in the treatment for human colon cancer.

关 键 词：短发卡状RNA RNA干扰 cerb—B1基因 表皮生长因子受体 结肠癌 

分 类 号：Q786[生物学—分子生物学]