摘要
目的:观察端粒酶逆转录酶显性负突变体(DN-hTERT)对乳腺癌细胞MCF7端粒酶活性的抑制作用。方法:将携带DN-hTERT基因的真核表达载体IRES2-EGFP空载体通过脂质体2000介导转入MCF7细胞,以G418进行稳定筛选,得到阳性克隆;倒置荧光显微镜观察转染细胞的生长情况;逆转录聚合酶链反应(RT-PCR)检测转染细胞hTERT mRNA的表达;TRAP-ELISA方法检测转染细胞端粒酶活性。结果:MCF7细胞转染DN-hTERT后生长受到抑制;转染DN-hTERT的MCF7细胞(MCF7/DN)hTERT mRNA表达升高;改进端粒重复序列扩增法(TRAP-ELISA)检测MCF7/DN细胞端粒酶活性与转染空载体MCF7细胞(MCF7/I)比较显著降低(P<0.05)。结论:DN-hTERT能特异性抑制MCF7细胞生长和端粒酶活性。
cancer cells MCF7 by ：To explore inhibition of telomerase activity and malignant phenotype on breast Dominant Negative human telomerase reverase transcriptase gene （DN-hTERT）. Methods： DN-hTERT eukaryotic expression vector DN-hTERT-IRES2-EGFP and empty vector （I） IRES2-EGFP were transfected into MCF7 by lipofectamine2000, after being selected by G418, positive clones were obtained; Tthe transfected cells growth was observed with inverted fluorescence microscope. The expression levels of hTERT mRNA of transfected cells were determined by reverse transcriptase polymerase chain reaction （RT-PCR）. Telomerase activity of transfected cells was measured byTRAP-ELISE. Results：The proliferation of MCF7/DN cells were inhibited by DN-hTERT transtec- tion; The expression levels of hTERT mRNA increased in MCF7/DN. Telomeric length was shorter in MCF7/DN than that in MCF7/I. The telomerase activity measured by TRAP-ELISE was 2.36 ＋0.12 in MCF7/DN and was 3.32 ± 0.14 in vacant vector MCF7/I. The telomerase activity in MCF7/DN was significantly lower than in vacant vector transfected MCF7/I （ P 〈 0.05 ）. Conclusion ： DN-hTERT could inhibit malignant phenotype and telomerase activity on MCF7 cells.
出处
《医学研究生学报》
CAS
2007年第7期678-681,785,共5页
Journal of Medical Postgraduates
基金
国家自然科学基金资助项目(批准号:30500596)
关键词
人端粒酶逆转录酶显性负突变体
端粒酶活性
乳腺癌
Dominant negative human telomerase reverase transcriptase gene
Telomerase activity
Breast cancer
