摘要
目的:克隆人端粒酶逆转录酶启动子(Human telomerase reverse transcriptase,hTERT),探讨对乳腺癌细胞的特异性[1],利用RNAi技术靶向沉默耐药乳腺癌细胞MCF-7/ADR乳腺癌耐药蛋白(Breast cancer resistance protein,BCRP)基因[2]的表达,观察BCRP的mRNA变化,BCRP蛋白的表达以及对阿霉素耐药性的变化[3]。方法:构建重组质粒(pGenesil-CMV-shRNA、pGenesil-hTERT-shRNA),分别转染端粒酶阴性及阳性细胞,检测hTERT启动子的靶向性,应用RT-PCR及Western-blot技术检测不同组的细胞的RNA、蛋白差异,MTT法分析药敏性变化[4]。结果:端粒酶阳性的细胞可见很强的荧光,明显强于端粒酶阴性的细胞[5];细胞实验RT-PCR及Western blot检测结果显示:BCRP的mRNA和蛋白的表达受抑制与对照组其差异有显著性(P<0.05);MTT比色法结果显示:对阿霉素的药物敏感性明显提高。结论:使用hTERT启动子联合RNAi成功靶向沉默BCRP基因,乳腺癌MCF-7/ADR对阿霉素的敏感性明显增加,为进一步开发肿瘤的特异性基因沉默治疗奠定实验基础。
Objective：To clone the promoter of human telomerase reverse transcriptase （hTERT）and investigate its selective effect on breast cancer cell,and to observe changes of breast cancer resistance protein（BCRP） mRNA,its protein expression and changes of drug resistance to doxorubicin with RNAi technology used in cell （MCF-7/ADR）. Methods：Plasmids （pGenesil-CMV-shRNA,pGenesil-hTERT-shRNA）were recombined and transfected into telomerase-negative and-positive cells,hTERT promoter targeting was detected.RT-PCR ,and Western-blot were applied to detect RNA and protein of different groups of cells. MTT was applied to analyze drug sensitivity. Results：Highly visible fluorescent was seen in telomerase-positive cells ,which was stronger than that of telom-erase-negative cells.The results of RT-PCR , and Western-blot showed that the mRNA and protein expression of BCRP were inhibited ; MTT results showed that the drug sensitivity to doxorubicin was significantly increased , which was of significant difference as compared with the control group （ P0.05 ） . Conclusion ： hTERT promoter can targeting silence BCRP gene combined with RNAi technology and drug resistance to doxorubicin is reversed,which lays a solid experimental basis for the further development of tumor specific therapy in gene silencing.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2010年第10期1482-1485,共4页
Journal of Chongqing Medical University
基金
重庆市教委科学技术(编号:KJ080305)
