hTERT基因转染对人胚胎成纤维细胞表型的影响

Effects of hTERT gene transfection on the phenotypes of human embryonic fibroblasts

目的 探讨外源性人端粒酶蛋白催化亚单位 (hTERT)基因转染人胚胎成纤维细胞 (hEFs)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分别检测hTERTmRNA和蛋白质的表达。采用染色体核型分析、裸鼠皮下致瘤性实验、DNA倍体实验分析转染细胞是否存在恶性表型。结果 原代培养hEFs中存在较弱水平的hTERT基因mRNA及蛋白质表达 ,但外源性hTERT基因转染的胚胎成纤维细胞hTERTmRNA及蛋白表达显著增加。hTERT转染后细胞染色体仍为 2 3对 ,且均为正常二倍体细胞 ;裸鼠皮下不具有成瘤的能力。

Objective To explore the effects of exogenous human telomerase reverse transcriptase (hTERT) on the phenotypes of human embryonic fibroblasts (hEFs). Methods Sense recombinant plasmid pIRES2 EGFP hTERT and empty vector plasmid pIRES2 EGFP were transfected into primary cultured hEFs by lipofectin reagent. Then hTERT expression and protein expression were analyzed respectively by Western blotting. Tumorigenecity in nude mice was analyzed and chromosome karyotype and cell DNA ploidy analyses were performed in the transfected and untransfected cells. Results Low level of hTERT expression was found in the primary cultured hEFs. Exogenous hTERT expression of hEFs increased significantly after hTERT transfection. Moreover, there were still 46 chromosomes in hTERT transfected hEFs without abnormal DNA ploidy. No tumorigenecity was found in the nude mice. Conclusion There are no malignant phenotypes in hTERT transfected hEFs.