DN-hTERT对MCF7细胞端粒酶活性及细胞生长的抑制作用

Inhibition of Telomerase Activity and Malignant Phenotype by DN-hTERT in Breast Cancer Cells

目的:观察端粒酶逆转录酶负突变体(DN-hTERT)对乳腺癌细胞MCF7端粒酶活性和恶性表型的抑制作用。方法:将带DN-hTERT基因的真核表达载体(DN-hTERT-IRES2-EGFP,DN)、IRES2-EGFP空载体(Ⅰ)通过脂质体2000(lipofectamine2000)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;流式荧光原位杂交法(FLOW-FISH)检测转染细胞端粒长度的变化;裸鼠肿瘤细胞移植观察转染细胞动物致瘤率。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;TRAP-ELISA检测MCF7/DN细胞端粒酶活性(2.36±0.12)与转染空载体MCF7细胞(MCF7/I)(3.32±0.14)比较显著降低(P<0.05);反应端粒长度的荧光强度差比较中,MCF7/DN细胞(13.89±0.23)比MCF7/I(19.87±0.36)低,转染DN端粒长度出现缩短;裸鼠肿瘤细胞移植,2周动物致瘤率MCF7/DN为0,与MCF7/I的35%比较有极显著意义(P<0.01)。结论:DN-hTERT能特异性抑制MCF7细胞生长和端粒酶活性。

Objective： To explore the inhibition of telomerase activily and malignant phenotype of tile Dominant Negative human telomerase reverse transeriptase gene （DN-hTERT） transfected into MCF7 breast cancer cells. Methods： DN-hTERT eukaryotie expression vector DN-hTERT-IRES2- EGFP and empty vector IRES2-EGFP were transfected into MCF7 cells using lipofeetamine2000. After selection with G418, positive clones were obtained. Growth of the transfeeted cells was observed with inverted fluorescence microscopy. The expression levels of hTERT mRNA in the lransfected cells was determined by reverse transeriptase polymerase chain reaction （RT-PCR）. Telomerase activity in transfeeted cells was measured by TRAP-ELISA. Telomeric length was measured by FLOW-FISH. The tumorigenic potential of transfeeted cells was observed in nude mice. Results： The proliferation of MCF7 cells transfecled with DN-hTERT-IRES2-EGFP （MCF7/DN） was inhibited. The expression levels of hTERT mRNA increased in MCF7/DN cells. Telomeric length was shorter in MCF7/DN cells than in MCF7 cells transfected with emply vector （MCF7/I）. The telomerase activity measured by TRAP-ELISA in MCF7/DN cells was 2.36±0.12 and in MCF7/I it was 3.32±0.14, a difference that is statistically significant （P〈0.05）. The tumorigenic ratio in nude mice was 0% tot MCF7/DN cells and 35% for MCF7/I cells after 2 weeks. There was a significant difference between the ratios for MCF7/ DN and MCF7/I （P〈0.01）. Conclusion： DN-hTERT inhibits malignant phenotype and telomerase activity in MCF7 cells.