人脐血间充质干细胞体外分离培养及基因载体特性研究

Study on isolation and culture of mesenchymal stem cells in human umbilical cord blood and the using of MSCs as gene vector

目的证实脐血中含有间充质干细胞,探讨其体外分离、培养条件,并进行生物学特性和表面抗原的鉴定,初步探索间充质干细胞作为基因治疗的载体携带目的基因的可能性及方法。方法无菌条件下取正常足月剖宫产的脐血分离、培养和纯化,获得间充质干细胞;对获得的间充质干细胞进行形态学观察;绘制生长曲线,分析间充质干细胞的增殖情况;用流式细胞仪检测间充质干细胞表面抗原的表达情况。采用脂质体转染法将pDNA316-IRES-EGFP质粒转入间充质干细胞中,观察绿色荧光的表达以及转染后细胞生长情况。结果自脐血中成功分离获得间充质干细胞,可传代培养,经表面标志检测,表达CD29、CD44、CD105,不表达CD45和HLA-DR。质粒pDNA316-IRES-EGFP通过脂质体介导转染间充质干细胞后,荧光显微镜下观察到报告基因EGFP的表达。转染后的细胞生长受到一定影响。结论人脐血来源的间充质干细胞能在体外分离、培养、扩增,并且具有和骨髓源间充质干细胞类似的生物形态和抗原表型。EGFP基因可被成功转入间充质干细胞中并获表达。转基因间充质干细胞用于基因治疗的持久性和有效性仍需进一步研究。

Objective：To investigate there are mesenchymal stem cells（MSCs） in human umbilical cord blood（HUCB）,and identify the biological properties and surface antigens of MSCs.To explore the possibility and method of MSCs carrying EGFP gene as a vector.Methods：MSCs were isolated and cultured from HUCB with density gradient centrifugation,observed their morphology,drawn growth curve,and detected the surface antigens using the flow cytometer.pDNA316-IRES-EGFP containing EGFP as report gene was introduced by lipofectamine transfection reagent into MSCs.The expression of EGFP was detected by fluorescent microscope.Result：MSCs had been isolated from HUCB successfully,and could be cultured stably in vitro.MSCs expressed CD29,CD44 and CD105,but did not expresse CD45 and HLA-DR.pDNA316-IRES-EGFP had been introduced by lipofectamine transfection reagent into MSCs.Green fluorescence protein in MSCs had been observed by fluorescent microscope.The growth of MSCs was inhibited after gene transfection.Conclusions：HUCB is one of the source of MSCs.Exogenous EGFP gene can be successfully transferred into MSCs and obtain permanent expression.Further researches are needed in MSCs for gene therapy.