骨髓间充质干细胞向功能性神经元的横向分化

Trans-differentiation of bone marrow mesenchymal stem cells into functional neurons

背景:骨髓间充质干细胞定向分化为神经干细胞并探讨提高分化效率的方法,具有重要的理论与实际应用意义。目的:建立以不同诱导方法横向分化大鼠骨髓间充质干细胞为功能神经元的方法。方法:分离、纯化得到骨髓间充质干细胞,流式细胞仪检测骨髓间充质干细胞表面标志物。将骨髓间充质干细胞分为神经营养因子诱导组、化学诱导组和对照组,骨髓间充质干细胞分别加入脑源性神经营养因子和碱性成纤维细胞生长因子、二甲亚砜和丁香茴醚、PBS进行诱导。结果与结论:神经营养因子诱导组和化学诱导组诱导后的细胞均表达神经元特异性烯醇化酶和微管相关蛋白2,且2组细胞的表达量接近(P>0.05),而对照组未发现神经元特异性烯醇化酶和微管相关蛋白2阳性表达。膜片钳系统检测发现神经营养因子诱导组诱导后的细胞具有神经细胞特征性的动作电位和兴奋性突触后电流,而化学诱导组和对照组均未发现动作电位和兴奋性突触后电流。提示脑源性神经营养因子联合碱性成纤维细胞生长因子是一种诱导骨髓间充质干细胞横向分化为功能神经元的可靠方法。

BACKGROUND： It has the important theoretical and practical significance to induce the differentiation of bone marrow mesenchymal stem cells into neural stem cells and to investigate the method to improve the differentiation efficiency. OBJECTIVE： To investigate the method of trans-differentiation of bone mesenchymal stem cells into functiona neurons. METHODS： Bone marrow mesenchymal stem cells were isolated and purified from rat bone marrow by density gradient centrifugation and adherent culture. Expression of bone marrow mesenchymal stem cells surface marker was detected by flow cytometry. All bone marrow mesenchymal stem cells were divided into three groups： neurotrophic factor induction group, chemical induction group and control group. The bone marrow mesenchymal stem cells in the neurotrophic factor induction group were induced with brain-derived neurotrophic factor and basic fibroblast growth factor, the cells in the chemical induction group were induced with dimethyl sulfoxide and butylated hydrochloride, and the cells in the control group were induced with PBS. RESULTS AND CONCLUSION： The bone marrow mesenchymal stem cells in the induction groups could express neuron specific enolase and microtubule-associated protein-2, and there was no significant difference of the expression rate between these two groups （P 〉 0.05）, while in the control group, no expression of neuron specific enolase and microtubule-associated protein-2 was detected. Patch clamp system detection showed that the induced bone marrow mesenchymal stem cells in the neurotrophic factor induction group could express action potential with the characteristics of neural cells and excitatory postsynaptic currents. There were no electrophysiologica characteristics in the chemical induction group and control group. It indicates that brain-derived.neurotrophic factor combined with basic fibrobiast growth factor is an effective method of inducing bone marrow mesenchymal stem cells to trans-differentiate into functional neurons.