bmi-1shRNA逆转录病毒表达载体的构建及稳定转染LoVo细胞系的建立  

Construction of Retrovirus-mediated shRNA Expression Vector Targeting bmi-1 and Establishment of Stable Transfected LoVo Cell Line

作　　者：汪云霞[1] 魏亚明[2] 王晓华[1] 朱晓莹[1] 黄颖烽[2] 

机构地区：[1]广州医学院生化教研室,510182 [2]广州医学院附属广州市第一人民医院中心实验室广州市临床医学研究所

出　　处：《肿瘤防治研究》2010年第3期269-273,共5页Cancer Research on Prevention and Treatment

基　　金：广州市科技计划资助项目(2007J1-C0091;2008J1-C0171)

摘　　要：目的构建针对原癌基因bmi-1的shRNA逆转录病毒表达载体,建立稳定转染的LoVo细胞系,探讨稳定转染bmi-1shRNA对大肠癌细胞LoVo靶基因表达的影响,为下一步应用RNAi技术治疗打下基础。方法设计合成靶向bmi-1基因编码短发夹RNA的两条寡核苷酸序列,另设计无义对照序列,构建重组载体pSIREN-bmi-1及pSIREN-bmi-1-C,载体经脂质体2000介导转染LoVo细胞,通过嘌呤霉素筛选,建立稳定转染的LoVo细胞系,显微镜观察、MTT检测、荧光定量PCR、Western blot检测bmi-1表达受抑后对细胞的增殖影响。结果成功构建了针对bmi-1基因的shRNA表达载体,建立了稳定转染的LoVo细胞系,bmi-1shRNA对LoVo细胞bmi-1mRNA表达抑制率为80%、bmi-1蛋白抑制率为82%,P<0.05。结论针对bmi-1基因的shRNA表达载体能够明显抑制结肠癌细胞LoVo的增殖能力,bmi-1mRNA与蛋白表达受抑制,但对细胞形态学无明显影响。Objective To construct Retrovirus-mediated shRNA expression vector targeting bmi- 1, which could transfect LoVo cells so as to establish stable LoVo cell line and to investigate the effect of stable expression brni-lshRNA in LoVo cells, paving the way for further application in RNAi gene therapy. Methods According to the principles of RNA interference technology, we designed and constructed the complementary oligonucleotides encoding hairpin RNA which specifically targets bmi-1 gene. The nonsense oligonucleotides were also designed and constructed. Then, we transfected them into colon cancer LoVo cells by lipofectamineTM 2000. After screening culture by puromycin, stable transfected LoVo cell line was established. We observed the proliferationchange of stable transfected LoVo cell line after the inhibition of bmi-1 by microscope, MTT, fluorescence quantitative PCR and Western blot. Results The vectors expressing shRNA targeting bmi-1 was constructed successfully. Stable transfected LoVo cell line was established. The rate of inhibition on bmi-1 mRNA is 80% and the rate of suppression on bmi-1 protein is 82%（P〈0. 05）. Conchtsion The vectors expressing shRNA targeting bmi-1 can inhibit cellular proliferation and bmi-1 gene expression, but the vectors had no obvious affects on cell morphology.

关 键 词：BMI-1基因 RNA干扰 结肠癌细胞 转染 短发夹RNA 

分 类 号：Q735.35[生物学—分子生物学]