端粒酶逆转录酶激活脐静脉内皮细胞端粒酶活性

Activation of telomerase activity of human umbilical vein endothelial cells by hTERT

目的 探讨外源性端粒酶逆转录酶 (hTERT)能否激活人脐静脉内皮细胞端粒酶活性 ,为建立体外人脐静脉内皮细胞系奠定基础。方法 用逆转录病毒转染法 ,将编码hTERT片段逆转录病毒载体与VSV G共转染 2 93 GP2 细胞 ,产生病毒上清液 ,感染原代分离的人脐静脉内皮细胞 (hUVEC) ,以嘌呤霉素筛选。观察细胞生长曲线、第Ⅷ因子、CD34、β 半乳糖苷酶染色、逆转录 聚合酶链反应 (RT PCR)等指标。 结果 细胞生长曲线显示培养至 30代的转染细胞生长速度基本与原代培养脐静脉内皮细胞一致但快于原代培养传至第 10代内皮细胞 ;RT PCR结果转染细胞有特异扩增 ;衰老指标 β 半乳糖苷酶染色显示转染后细胞胞浆内着染阳性细胞数明显少于第 10代内皮细胞。结论 外源性hTERT转入原代培养人脐静脉内皮细胞 ,可重现其端粒酶活性 。

Objective To study whether the telomerase activity can be activated by human telomerase reverse transcriptase (hTERT)and hTERT is expressed in human umbilical vein endothelial cells (hUVECs).Methods The retrovirus vector with hTERT and VSV-G were Used to co-transfect 293-GP 2 cell line, hUVECs were infected by retrovirus supernatant and selected with puromycin. Expression of hTERT gene was examined by RT-PCR,flow cytometry, senescence-associated SA-β-qalactosidase staining, survival curve and cell-cycle analysis.Results The presence of hTERT RNA transcribed from the transduced retroviral vector was confirmed by RT-PCR.hTERT DNA was expressed in genome of hUVEC cells. hUVECs with hTERT had a few or no SA-β-galactosidase positive oells.Conclusions hTERT was expressed in hUVECs by stable transfection and telomerase was activated by exogenous hTERT,thus preventing the senescent phenotype of hUVECs by hTERT expression. This is the first step to establish an immortal hUVECs line.