质粒介导的RNAi抑制端粒酶活性  

Suppression of Telomerase Activity by Plasmid-mediated RNA Interference

作　　者：李燕[1] 李明远[1] 彭颖 蒋中华[1] 李婉宜[1] 李虹[1] 

机构地区：[1]四川大学华西基础医学与法医学院病原生物学教研室,成都610041 [2]四川维奥制药有限公司,成都610041

出　　处：《生物医学工程学杂志》2006年第3期615-619,共5页Journal of Biomedical Engineering

基　　金：教育部博士学科点基金项目(20040610050);四川省科技厅基金资助项目(03JY-029-075-2)

摘　　要：为了探讨靶向人端粒酶反转录酶(hTERT)基因的小干扰RNA(s iRNA)表达载体是否具有抑制H eL a细胞端粒酶活性的能力,人工合成2条64个核苷酸(n t)的片段,其中19n t与hTERT基因1789-1807位碱基同源,将其退火、连接到质粒pSU PER中,构建pSU P-hTE。在pSU P-hTE基础上,把该质粒的启动子和64n t插入片段酶切、克隆到质粒pEGFP-C 1中生成pEGFP-hTE,从而获得新霉素抗性筛选质粒。将pEGFP-hTE用脂质体转染H eL a细胞,G 418筛选后获得抗性克隆并将其收获、传代,用不同方法检测hTERT的mRNA和蛋白表达水平、H eL a细胞的端粒酶活性以及细胞的增殖能力。结果显示,pEGFP-hTE转染的H eL a细胞与对照组比较,hTERT的mRNA水平下降及蛋白表达减少、细胞端粒酶活性降低38%,但细胞增殖能力没有明显改变。以上结果表明,pEGFP-hTE能通过RNA干扰(RNA i)途径特异性抑制H eL a细胞hTERT基因的表达,从而有效抑制细胞端粒酶活性,这可能将为肿瘤生物治疗提供一条新的途径。This study was aimed to construct a plasmid expressing siRNA specific for the human telomerase reverse transcriptase （hTERT） gene and to evaluate the ability of small interference RNA（sIRNA） for inhibiting telomerase activity in HeLa cells. 64 nucleotides, in which 19nt were homologous with hTERT gene, were chemically synthesized, annealed and linked into pSUPER to get pSUP-hTE. Then pSUP-hTE was digested with enzyme. We obtained its fragmant concluding promoter and 64nt. So we cloned it into pEGFP-C1 for constructing pEGFP-hTE which contains ned gene and the enhanced green fluorescent protein （EGFP）. Recombinant pEGFP- hTE was transfected to HeLa cells. These cells were screened with medium containing G418. When stable colonies appeared, G418-resistant cells were harvested and propagated. At the different cell generations, hTERT mRNA and protein expression, telomerase activity and cell growth activity were analyzed. Compared with control cells, HeLa cells transfected with pEGFP-hTE showed that hTERT mRNA level and hTERT protein expression decreased and telomerase activity reduced by 38%, but the cells growth activity displayed no changes. So pEGFP- hTE could specifically inhibit expression of hTERT and telomerase activity. These results suggested that sIRNA targeting hTERT gene might provide a new strategy for cancer biotherapy.

关 键 词：PSUPER RNA干扰 人端粒酶区转录酶 抑制端粒酶活性 

分 类 号：R346[医药卫生—基础医学]