外胚间充质干细胞永生化的实验研究

Immortalization of ectomesenchymal stem cells by transfecting hTERT gene

目的 :探讨端粒酶催化亚基 (hTERT)在外胚间充质干细胞永生化中的作用。方法 :质粒pCI neo -hTERT转染外胚间充质干细胞 ,经G4 18筛选后获得抗性克隆。免疫细胞化学法及图像分析检测hTERT的表达 ,累计生长曲线检测细胞增殖能力。结果 :转染后获 3个抗性克隆。转染第 3代细胞hTERT染色呈强阳性 ,转染后第 15代细胞hTERT活性较正常培养的第 15代细胞明显增强。转染细胞增殖能力有不同程度的增加 ,其中克隆 2越过衰老期 ,寿命延长。结论 :外源性hTERT介入可重建外胚间充质干细胞的端粒酶活性 ,使细胞延长寿命 ,得以永生。

AIM: To investigate the effect of hTERT in the immortalization of ectomesenchymal stem cell. METHODS: Ectomesenchymal stem cells were transfected with eurayotic expressing plasmid pCIneo-hTERT and encoding the hTERT.Immunohistochemistry assay was applied to identify the hTERT activity of anti-G418 clones.Growth curve was tested to evaluate the proliferating ability of these clones. RESULTS: 3 anti-G418 clones were obtained.After transfecting,the 3 rd passage cells expressed hTERT strongly.The 15 th passage cells expressed hTERT much stronger than the 15 th passage cell without transfecting hTERT.After transfecting,proliferating ability were increased in certain extent. Clone 2 had maintained in culture and overcome senescence. CONCLUSION: Ectomesenchymal stem cells could overcome senescence and immortalized by transfecting hTERT.