过表达人端粒酶逆转录酶促进人脐静脉血管内皮细胞增殖

Overexpression of hTERT Promotes the Proliferation of Human Umbilical Vein Endothelial Cells

人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)是端粒酶的催化亚基,专司在染色体末端添加端粒重复序列,与维持端粒长度与功能有关.为探索hTERT的生物学功能,本研究以人脐静脉血管内皮细胞(hUVEC)为研究对象,观察了hTERT基因过表达对hUVEC细胞增殖作用和端粒酶活性的影响.利用构建的pEGFP-C1-hTERT融合基因表达载体转染hUVEC,结果显示,转染后的细胞可见GFP表达,提示转染细胞有GFP-hTERT融合蛋白表达.RT-PCR、免疫组化和TRAP-PCR-ELISA法显示,与未转染细胞比较,转染细胞的hTERT mRNA、蛋白质表达水平明显升高,端粒酶活性明显增强.MTT实验显示,转染hTERT基因的细胞在72 h后细胞增殖速率明显大于未转染细胞及空载体转染细胞.结果提示,过表达hTERT基因可提高血管内皮细胞端粒酶的活性和增殖能力.实验结果证明,端粒酶活性与细胞增殖活性密切相关.

Human telomerase reverse transcriptase（hTERT） is the catalytic subunit of the telomerase,which is responsible for addition of TTAGGG repeats to the telomere ends of the chromosome.In order to investigate the main functions of hTERT gene,we applied gene transfer technique to promote the overexpression of extrinsic hTERT gene in human umbilical vein endothelial cells（hUVECs） and observed the effects on cell proliferation and telomerase activity.The eukaryotic expression vector pEGFP-C1-hTERT was constructed with PCI-neo-hTERT and pEGFP-C1 plasmids,and the accuracy of hTERT gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.Then theplasmid was transfected into hUVECs by liposome-mediated transfection.The results indicated that thereporter gene GFP was expressed after transfection.RT-PCR,immunocytochemical assay（ICA）andTRAP-PCR-ELISA showed that hTERT mRNA,protein expression and telomerase activity wereremarkably increased in transfected hUVECs.The cells transfected with pEGFP-C1-hTERT（hUVEC-hTERT）grow faster than the cells transfected with pEGFP-C1（hUVEC-Null）and control（hUVEC）cells after 72 hours.Overexpression of hTERT gene promotes the telomerase activity and cell proliferationof hUVECs.These preliminary data confirm that telomerase activity and cell proliferation are closely related.