端粒酶表达延长人骨髓间充质干细胞生命周期的实验

Experiment of telomerase expression extending life span of human bone marrow der ived mesenchymal stem cells

目的:构建并鉴定人端粒酶反转录酶(humantelomerasereversetranscrip-tase,hTERT)和增强型绿色荧光蛋白EGFP)双基因共表达重组真核表(达质粒并转染人骨髓间充质干细胞(humanbonemarrowderivedmes-enchymalstemcells,hBMMSCs)。比较转染细胞与非转染细胞体外培养的生命周期。方法:实验于2002-10/2003-06在第三军医大学医院烧伤研究所完成。扩增pGRN145质粒,EcoRⅠ酶切回收hTERT片断;利用基因重组技术将hTERT连接到IRES2-EGFP载体中,XhoⅠ酶切和测序分别对重组质粒进行鉴定。用NucleofectorTM技术将构建的重组质粒转染hBMM-SCs。相同条件下体外培养转染细胞与非转染细胞,分别观察其细胞形态和生命周期。结果:成功构建了hTERT和EGFP双基因共表达重组真核表达质粒并成功转染了hBMMSCs。体外培养结果显示:非转染细胞到27PD(Populationdoubling)时出现细胞衰老、死亡、增殖停止;转染细胞到70PD时细胞形态无明显改变,细胞继续生长。结论:hTERT异位表达可以显著延长hBMMSCs体外培养生命周期,这为基因修饰hBMMSCs的单克隆筛选奠定了基础。

AIM:To construct and identify that human bone marrow derived mesenchymal stem cells were transduced by a recombinant eukaryotic expression plasmid containing both human telomerase reverse transcriptase(hTERT) and enhanced green fluorescen t protein(EGFP) genes.Then life spans in vitro of transduced cells and non tran sduced cells were compared. METHODS:The experiment was completed in the Institute of Burn,Third Military M edical University of Chinese PLA from October 2002 to June 2003.pGRN145 plasmid was amplified and cut by restriction endonuclease EcoRⅠ,then the hTERT segment was collected and recombined with eukaryotic expression vector IRES2 EGFP with gene recombination technique.The recombinant plasmid was identified by cutting o f restriction endonuclease XhoⅠand sequence determination and was transduced in to hBMMSCs with NucleofectorTM technique.Transduced cells were culture expended in vitro,and its life span and cell shape were observed and compared with non t ransduced hMSCs. RESULTS:The recombinant dicistronic eukaryotic expression plasmid of hTERT and EGFP genes has been constructed and tranfered into hBMMSCs succesfully.The tran sduced cells gained more than 70PD life span and had no visible change of cell s hape.Whereas non transduced hMSCs appeared cell senescence,cell death and proli feration suspend 001 at 27PD. CONCLUSION:Ectopic expression of hTERT in hBMMSCs can extend life span of hBM MSCs in vitro markedly.This work establishes the base of mono clonal screening of gene modified hBMMSCs.