生长因子诱导骨髓基质细胞向胆碱能神经元分化的研究

Study on Cholinergic Neuron Differentiation Induced by Growth Factors from BMSCs

目的建立生长因子定向诱导骨髓基质细胞(BMSCs)分化为胆碱能神经元的体系。方法 SD大鼠原代BMSCs体外培养,分别用神经生长因子(NGF)、NGF加碱性成纤维细胞生长因子(bFGF)、NGF加bFGF和表皮生长因子(EGF)诱导1、3、5 h,免疫荧光染色鉴定神经元表面标志微管联合蛋白-2(MAP-2)和胆碱能神经元标志物胆碱乙酰转移酶(ChAT),计数阳性细胞率。结果原代培养BMSCs传至第5代已基本纯化,NGF 100 ng/ml能有效诱导BMSCs分化为神经元,NGF、bFGF和EGF联合诱导可使BMSCs定向分化为胆碱能神经元,分化率为(67±8)%。结论 NGF、bFGF和EGF联合使用是诱导BMSCs定向分化为胆碱能神经元的有效组合,细胞分化率高。

Objective To screen and optimize the methods of bone marrow stromal cells（BMSCs） differentiation into cholinergic neurons induced by growth factors in vitro.Methods BMSCs culture and proliferation were established by using NGF with bFGF and EGF to induce BMSCs differentiation.Morphology of cell differentiation was observed and photographed by inverted phase contrast microscope to screen and determine the optimal concentration.Immunofluorescence identification of mitogen-activated protein 2（MAP-2）,ChAT expression and Proportion of ChAT-positive cells of induced BMSCs differentiation were used to select optimal combination of the treatments.Results BMSCs were purified at 5th generation.NGF with concentration of 100 ng/ml could induce BMSCs differentiation more effectively.Combination of NGF with bFGF and EGF showed a remarkable effect in inducing BMSCs differentiating into cholinergic neurons.Proportion of ChAT-positive cells were（67±8）%.Conclusion Combination of NGF with bFGF and EGF is an effective way in inducing BMSCs differentiation to cholinergic neurons with high following-protortion of differentiation.