增强型绿色荧光蛋白基因转入人骨髓间充质干细胞诱导其向神经元样细胞分化的示踪标记观察

Human bone marrow mesenchymal stem cell transducted by enhanced green fluorescent protein gene differentiated into neuron-like cells A trace observation

目的:建立携带示踪标记的人骨髓间充质干细胞,并将其向神经元样细胞定向诱导分化。方法:实验于2003-10/2005-01在郑州大学干细胞研究中心完成。用阳离子脂质体转染法将增强型绿色荧光蛋白基因转入人骨髓间充质干细胞中,然后用表皮生长因子和碱性成纤维生长因子联合诱导法将其向神经元样细胞诱导分化,并用反转录聚合酶链反应法对诱导后的细胞进行鉴定。结果:①转染48h后,在荧光显微镜下观察,对照组的人骨髓间充质干细胞不发荧光,而实验组的人骨髓间充质干细胞开始发荧光,经G418筛选后得到稳定表达增强型绿色荧光蛋白的人骨髓间充质干细胞。②转染后的人骨髓间充质干细胞经重组人表皮生长因子和碱性成纤维生长因子的联合诱导后,较多细胞出现了典型的神经元样形态,经反转录聚合酶链反应法证明,神经元特征性蛋白微管相关蛋白2和神经丝亚单位-M表达增强。结论:应用阳离子脂质体转染法将增强型绿色荧光蛋白基因转入人骨髓间充质干细胞中可获得稳定表达,转染后的人骨髓间充质干细胞,经表皮生长因子和碱性成纤维生长因子联合诱导后可以向神经元样细胞定向分化。

AIM：To establish human bone marrow mesenchymal stem cells （BMSCs） with trace labeling, then induce the cells to differentiate into neuron-like cells. METHODS： The experiment was conducted in the Stem Cell Research Center, Medical College of Zhengzhou University between October 2003 and January 2005. Enhanced green fluorescent protein gene was transducted into human bone marrow mesenchymal stem cells by lipesomemedicated transduction. The transducted BMSCs were cultured in vitro and induced into neuron-like cells with EGF and basic fibroblasts growth factor （bFGF）. The induced cells were identified with reverse transcriptionpolymerase chain reaction （RT-PCR）. RESULTS： ① At 48 hours after transfection, it was observed under fluorescence microscope that the BMSCs in the control group were not fluorescent, but those in the experimental group were fluorescent, and stable expressed human BMSCs of enhanced green fluorescent protein were selected with G418. ②After the induction of recombinant EGF combined with bFGF, the transfected BMSCs appeared typical neuron-like cells. RT- PCR analysis showed that the expressions of neuron-specific markers, such protein （MAP） and neurofilament subunit-M, were enhanced. CONCLUSION： Enhanced green fluorescent protein gene was transducted into human BMSCs by the technique of gene transduction and gained stable expression. The EGF and bFGF transducted BMSCs can differentiated into neuron-like cells.