端粒酶催化亚单位基因转染对人胚胎成纤维细胞表型及寿命的影响

The effects of hTERT transfection on the life-span and malignant phenotypes of human embroynic fibroblasts

目的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)寿命的影响以及基因转染后细胞是否存在恶性表型。方法应用脂质体法将pIRES2-EGFP-hTERT正义重组质粒及pIRES2-EGFP空载质粒分别导入原代培养hEFs,Westernblot检测hTERT蛋白及I、III型胶原表达。染色体核型分析、裸鼠皮下致瘤性实验、DNA倍体实验以及形态学观察分析转染细胞是否存在恶性表型。结果外源性hTERT基因转染的胚胎成纤维细胞hTERT蛋白和I、III型胶原表达增加,体外传代次数增加,传至75代仍保持以2d的倍增周期生长,并且染色体仍为23对,均为正常二倍体细胞;裸鼠皮下不具有成瘤的能力;而hEFs和空载转染细胞传至60代左右平均五六天传代1次,出现生长停滞现象。结论外源性hTERT基因转染使胎儿成纤维细胞增殖旺盛,寿命延长,且不具有恶性表型。

Objective The sense recombinant fluorescent e ukaryotic expressing vector containing hTERT cDNA was transfected into hEFs to explore the effects of exogenous h TERT on the life-span and malignant p henotypes of hEFs.Methods pIRES2-EGFP-hTERT and pIRES2-EGFP plasmid were transfected into primary hEFs by Lipofectin re agent.Then expression of hTERT,I an d III type collagen by Western blot,cell morphologhy under light telescope,tumorigenesis in nude mice,chromosome karyotype and cell DNA ploidy analysis were examined and evaluated in transfected and untransfected cells.Results The expression of exogenous hTERT an d I ,III type collagen of hEFs were increased by hTERT transfectio n.hTERT transfected hEFs showed lon ger population doubling time (PDs )than that of primary hEFs and hEFs transfected by vacant vectors.The hTERT transfected hEFs maintain ed to double within 2days until 75PDs ,while the growth of primary hEFs and hEFs transfected by vacant v ector turn to very slow at 60PDs.More over,the chromosome of hTERT transfected hEFs was still 46and displayed no abnormal DNA ploidy.No tumorigenicity was found in nude mice.Conclusions The life-span of hEFs could be extend ed by exogenous hTERT transfection,and t here is no malignant phenotypes in hT ERT transfected hEFs.