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
Cancer Research on Prevention and Treatment(2010)
Abstract
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。
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Key words
Transfection,Colorectal cancer,bmi-1,shRNA,RNA interference
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