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为了探讨外源性人端粒酶蛋白催化亚单位 (hTERT)基因转染对人胚胎成纤维细胞 (hEFs)端粒长度、端粒酶活性及其亚单位的影响 ,采用基因重组技术构建hTERT全长cDNA正义荧光真核表达载体 ,并采用脂质体法将正义重组质粒pIRES2 EGFP hTERT及空载质粒pIRES2 EGFP分别转染原代培养hEFs,检测转染细胞端粒长度、端粒酶活性及端粒酶亚单位的变化。结果显示 ,外源性hTERT基因转染细胞 (hEF hTERT)端粒酶活性较未转染细胞 (hEFs)及空载体转染细胞 (hEF EGFP)显著增加 (P <0 0 1) ,hEF hTERT、hEFs及hEF EGFP端粒长度分别为 6 0kb、5 3kb及 5 4kb ,端粒酶亚单位中除hTERT在mRNA和蛋白水平表达均增加外 ,hTR和TP1mRNA无明显变化。以上结果提示 ,外源性hTERT基因转染能使原代培养的hEFs端粒酶活化 ,端粒长度不再继续缩短 ,hTERT在mR NA和蛋白水平表达增加
In order to investigate the effect of exogenous hTERT gene transfection on telomere length, telomerase activity and its subunits in human embryonic fibroblasts (hEFs), hTERT was constructed by gene recombination The recombinant plasmid pIRES2 EGFP hTERT and empty vector pIRES2 EGFP were transfected into primary cultured hEFs by lipofectamine 2000 respectively. The telomere length, telomerase activity, Changes in granzyme subunit. The results showed that telomerase activity of exogenous hTERT gene transfected cells (hEF hTERT) was significantly higher than that of untransfected cells (hEFs) and transfected cells (hEF EGFP) (P <0.01), hEF hTERT, The telomere lengths of hEFs and hEF EGFP were 60kb, 53kb and 54kb, respectively. There were no significant changes in hTR and TP1mRNA in telomerase subunits except hTERT increased at mRNA and protein levels. The above results suggest that exogenous hTERT gene transfection can activate the primary cultured hEFs telomerase, telomere length no longer continue to shorten, hTERT mR NA and protein expression increased