外源性端粒酶催化亚单位对人视网膜血管内皮细胞的影响

Effect of Exogenously Expressed Telomerase Catalytic Subunit Gene Activity in Human Retinal Capillary Endothelial Cells

目的探讨外源性人端粒酶催化亚单位(hTERT)对人视网膜血管内皮细胞(hRCEC)端粒酶活性、相关基因表达和体外生存时间的影响。方法阳离子脂质体介导hRCEC转染;RT-PCR法检测人类端粒酶RNA组份(hTR)、端粒酶相关蛋白1(TEP1)及hTERT基因的表达;TRAP法检测端粒酶活性;细胞计数法绘制生长曲线。结果hTR、TEP1基因在hTERT转染hRCEC前后都表达,hTERT基因在转染前不表达,转染后24h及稳定转染克隆形成时表达,细胞停止分裂时又失去表达;转染前无端粒酶活性,转染后24h及稳定转染克隆形成时可检测到端粒酶活性,细胞停止分裂时端粒酶活性消失;稳定转染了hTERT基因后hRCEC的生存时间没有延长。结论hTERT转染可显著提高hRCEC中hTERT基因表达水平,hTERT基因表达与端粒酶活性一致,但只用hTERT转染的方法不能使hRCEC细胞生存时间延长。

Objective To examine the expression and activity of telomerase, and the life span of human retinal capillary.lary endothelial ceils （hRCEC） transfected with plasmid pCI-Neo-hTERT. Methods pCI-Neo-hTERT was transfected into hRCEC hy liposomes. Reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the genes expression of telomerase RNA component （hTR）,telomerasc associated protein1 （TEP1）and telomerase catalytic subunit （hTERT）. The telomerase activity of hRCEC was measured using telomeric repeat amplification protocol （TRAP） assay. The growth of hRCEC cells was determining by counting the cell number. Results Expression of hTR and TEP1 was seen in hRCEC before and after transfection. Before transfection, expression of hTERT was not observed in hRCEC. Telomerase activity was detected in cells at 24 hours after transfcetion. The life span of stablely transfectcd hRCEC was not extendcd. Conclusion Expression of tclomerase activity is closely associated with the expression of hTERT gene. Induction of telomerase could be achieved by transfecting the ceils with pCI-Neo-hTERT plasmid. However, it has no effect in prolonging the life span of hRCEC.