shRNA/hTERT表达载体靶向逆转乳腺癌的耐药性  

Targeting reversal of breast cancer drug resistance by BCRP shRNA expression vector with hTERT promoter in nude mice

作　　者：黄金向[1] 王继见[1] 

机构地区：[1]重庆医科大学附属第二医院普外科,重庆400010

出　　处：《第三军医大学学报》2011年第6期578-581,共4页Journal of Third Military Medical University

基　　金：重庆市教委资助项目(KJ080305)~~

摘　　要：目的克隆人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子,探讨对乳腺癌细胞的特异性,构建shRNA/hTERT表达载体,探讨在动物实验中耐药性的变化。方法构建重组质粒(pGenesil-CMV-shRNA、pGenesil-hTERT-shRNA),用MCF-7/ADR乳腺癌细胞接种于50只裸鼠前肢腋下,并将45只接种成功的动物模型按随机数字表法分为3组:对照组、重组质粒pGenesil-CMV-shRNA组、重组质粒pGenesil-hTERT-shRNA组,应用RT-PCR技术检测裸鼠组织的乳腺癌耐药蛋白(breast cancer resistance protein,BCRP)基因的mRNA及Western blot检测裸鼠组织BCRP基因的蛋白表达、测量裸鼠瘤体体积的变化。结果成功构建人乳腺癌裸鼠模型;动物实验RT-PCR及Western blot检测结果显示:pGenesil-CMV-shRNA、pGenesil-hTERT-shRNA组BCRP的mRNA和蛋白的表达均受抑制,与对照组比较,其差异均有统计学意义(P<0.05);裸鼠瘤体体积明显缩小,与对照组比较,其差异均有统计学意义(P<0.05);重组质粒pGene-sil-hTERT-shRNA组抑瘤率为68.5%。结论 shRNA/hTERT表达载体成功靶向沉默BCRP基因,动物实验证实对阿霉素的敏感性明显增加。Objective To clone human telomerase reverse transcriptase（hTERT） promoter and investigate its specificity to breast cancer cells,and to construct breast cancer resistance protein（BCRP） shRNA expression vector with hTERT promoter,pGenesil-hTERT-shRNA,and observe its influence on doxorubicin resistance of breast cancer cells in experimental animals.Methods Recombinant plasmids pGenesil-CMV-shRNA and pGenesil-hTERT-shRNA were constructed.Fifty nude mice were inoculated with human breast cancer cell line MCF-7/ADR.Forty-five tumor-carrying mice were evenly and randomly divided into a control group,a pGenesil-CMV-shRNA group and a pGenesil-hTERT-shRNA group according to random number tables.Western blotting and RT-PCR were applied to detect BCRP and its mRNA expression in mouse tissues.The tumor volume changes of the tumor-carrying mice were measured.Results The mouse model of human breast cancer was constructed.Western blotting and RT-PCR results showed that,in the pGenesil-CMV-shRNA and pGenesil-hTERT-shRNA groups,the expression levels of BCRP and its mRNA as well as the tumor volumes were significantly reduced as compared with those of the control group（P0.05）.The tumor inhibition rate of pGenesil-hTERT-shRNA groups was 68.5%,which were significantly higher than that of the control group（P0.05）.Conclusion BCRP shRNA expression vector with hTERT promoter can specifically silence BCRP gene and increase breast cancer cell susceptibility to doxorubicin.

关 键 词：重组质粒 启动子hTERT SHRNA MCF-7/ADR 端粒酶阳性细胞 裸鼠模型 

分 类 号：R394-33[医药卫生—医学遗传学] R73-361[医药卫生—基础医学]