神经生长因子转染骨髓间充质干细胞诱导分化成神经样细胞

Mesenchymal stem cells of bone marrow differentiate into neuron-like cells after transfected by nerve growth factor gene in vitro

目的探讨神经生长因子(NGF)转染大鼠骨髓间充质干细胞(MSCs)后的表达及其对MSCs分化成神经样细胞的影响。方法在体外低密度扩增大鼠骨髓MSCs。应用基因重组技术,构建pEGFP-NGF真核表达质粒,并将其转染至MSCs中。免疫荧光标记法检测中间神经丝蛋白(NF-M)和胶质纤维酸性蛋白(GFAP)的表达。在荧光显微镜下观察MSCs阳性荧光的表达率、细胞的形态变化和类型。结果MSCs阳性荧光的表达率约为30%。转染后,MSCs呈现多突起,且几个突起之间互相连接成网状,似神经元样的形态。免疫荧光标记法鉴定其中一部分细胞表达NF-M,另一部分细胞表达GFAP。结论转染的MSCs可表达NGF并在含有NGF的微环境中分化成神经样细胞。

Objective To study the gene expression of nerve growth factor （NGF） in rat bone marrow mesenchymal stem cells （MSCs）and the effect of NGF-GFP fusion protein on MSCs differentiation into neuron-like ceils. Methods Rat bone marrow mesenchymal stem cells were amplificated in low density in vitro. The NGF gene was inserted into pEGFP-N2C-terminal protein fusion vector to construct pEGFP-NGF, which was transfected into MSCs using Lipofectamine 2000. The infected efficiency, morphological change of MSCs and cell types were examined by fluorescent microscope. Results Infected efficiency of NGF-GFP fusion protein was 30% in MSCs. The transfected MSCs displayed neuron-like morphology and formed net. Some transfected MSCs expressed neurofilament protein-M （NF-M）, while the others was immuostained by glial fibrillary acidic protein （GFAP）. Conclusion MSCs might be used as carrier cells of gene therapy because they express NGF and differentiate into neuron-like cells in the environment with NGF.