靶向hTERT基因表达载体的构建和其对人乳腺癌细胞端粒酶活性及细胞增殖的影响  

Construction of an expression vector carrying short hairpin RNA targeting hTERT gene and its effects on breast cancer cell telomerase activity and proliferation in vivo

作　　者：刘祥厦[1] 姚陈[2] 张辉[3] 王三明[2] 王深明[2] 

机构地区：[1]中山大学附属第一医院整形外科,广东广州510080 [2]中山大学附属第一医院乳腺外科,广东广州510080 [3]中山大学附属第一医院麻醉科,广东广州510080

出　　处：《南方医科大学学报》2009年第11期2187-2190,共4页Journal of Southern Medical University

基　　金：广东省自然科学基金(021907)

摘　　要：目的构建靶向端粒酶hTERT基因mRNA的shRNA质粒表达载体,转染人乳腺癌MCF-7和MDA-MB231细胞系,探讨其对细胞端粒酶活性及细胞增殖的影响。方法设计合成端粒酶hTERT基因特异性shRNA干扰序列,重组pSuper-retro-puro质粒,转染乳腺癌MCF-7和MDA-MB231细胞系(转染组),设立正常培养乳腺癌MCF-7和MDA-MB231细胞系(阴性对照组)和pSuper-retro-puro质粒转染的乳腺癌MCF-7和MDA-MB231细胞系(空白对照组),利用端粒重复序列扩增-酶联免疫吸附测定法(TRAP-ELISA)检测细胞端粒酶活性,MTT实验及琼脂糖细胞集落形成实验检测细胞增殖能力等。结果成功构建pSuper-retro-puro-TERTRNAi#1、#2质粒并转染乳腺癌MCF-7和MDA-MB231细胞系,转染后细胞端粒酶活性较转染前明显下调,差异有统计学意义(P<0.005);MTT法检测细胞增殖能力显示,MCF-7及MDA-MB231转染组细胞增殖活性较阴性对照组低,差异有统计学意义(P<0.05);软琼脂集落形成实验表明MCF-7和MDA-MB231转染组细胞克隆形成能力较阴性对照组显著下降,差异有统计学意义(P<0.001)。结论通过RNAi技术特异性干扰hTERT基因mRNA表达可显著下调细胞端粒酶活性,并可导致细胞增殖能力的减弱,以端粒酶为靶点的基因治疗可能是乳腺癌治疗的一种有效方法。Objective To construct a RNA interference expression vector targeting human telomerase reverse transcriptase gene（hTERT） gene and investigate its effects on telomerase activity and proliferation in breast cancer cells in vitro.Methods The shRNA sequences targeting hTERT gene were designed and recombined into pSuper-retro-puro vector.The breast cancer cell lines MCF-7 and MDA-MB231 were transfected with the recombined vector,and the telomerase activity of the cells was tested by telomerase repeat sequence amplification-enzyme linked immunosorbent assay（TRAP-ELISA）.The proliferation of the transfected cells was assessed using MTT and soft-agar clone formation assays.Results The recombinant plasmids pSuper-retro-puro-TERT RNAi#1 and #2 were successfully constructed as confirmed by enzymatic digestion and DNA sequencing.The telomerase activity in the transfected breast cancer cells were down-regulated significantly as compared with that in negative control cells（P〈0.005）.The transfection resulted in significant inhibition of the proliferation of both MCF-7 and MDA-MB231 cells as detected by MTT assay（P〈0.05） and soft agar clone formation assay（P〈0.001）.Conclusion Transfection with the recombinant plasmid containing the shRNA targeting hTERT gene can down-regulate telomerase activity and inhibit proliferation of breast cancer cells in vitro,suggesting the potential of gene therapy targeting telomerase in the treatment of breast cancer.

关 键 词：乳腺癌 端粒酶 HTERT RNA干扰 基因治疗 

分 类 号：R737.9[医药卫生—肿瘤]