体外诱导骨髓间充质干细胞向神经元样细胞分化的研究

目的观察人骨髓间充质干细胞（BM-MSCs）体外扩增和定向诱导分化为神经元样细胞的变化,为其临床应用奠定基础。方法采用密度梯度离心法分离BM-MSCs细胞,用MesencultTM培养基和贴壁培养法培养、纯化和扩增细胞,流式细胞术检测细胞表面CD29和CD90分子表达率。采用20 ng/ml重组碱性成纤维细胞生长因子（bFGF）、20 ng/ml新型人表皮生长因子（hEGF）和20 g/L二甲基亚砜（DMSO）、5 mmol/Lβ-巯基乙醇（BME）分别诱导BM-MSCs;采用免疫组化法检测细胞的神经元特异性烯醇化酶（NSE）、胶原纤维酸性蛋白（GFAP）、波形蛋白（VIM）表达情况。结果BM-MSCs细胞增殖迅速,3周可传代培养;BM-MSCs传十代扩增1－2×10^3倍。分离和扩增BM-MSCs CD29、CD90强表达率分别为95.02%、93.81%。药物诱导后的BM-MSCs发生轴突等神经元样细胞变化,阳性表达NSE、GFAP、VIM分子。结论人BM-MSCs能体外培养和扩增,并能定向诱导分化为神经元样细胞。

Study on the differentiation into neuron-like cells of human bone marrow mesenchymal stem cells in vitro

Objective To observe the proliferation of human bone marrow mesenchymal stem cells（BM-MSCs） in vitro and directional-induced differentiation into neuron-like cells,so as to provide basis for its clinical application.Methods Density gradient centrifugation was used to separate BM-MSCs,MesencultTM culture medium and pasting-wall culture method were used to culture,sublimate and proliferate BM-MSCs,CD29and CD90 clusters expressions were tested by Flow cytometry.BM-MSCs were directionally induced by 20 ng/ml human basic fibroblast growth factor（bFGF）,20 ng/ml human epidermal growth factor（hEG F）,20 ng/ml dimethyl sulphoxide（DMSO） and 5 mmol/L β-mercaptoethanol（BME）.Immunohistochemistry was used to test the expression of neuron specific enolase（NSE）,glial fibrillay acidic protein（GFAP） and vimentin（VIM）.Results BM-MSCs proliferated rapidly,could be sub-cultured in 3 weeks.BM-MSCs could expand（1～2）×10^3 folds afer 10 times sub-culture.Separated and proliferated BM-MSCs expressed CD29 and CD90 clusters with the positive rate of 95.02% and 93.81% separately.The BM-MSCs occured some morphology changes included axon like structure after the induction of drugs,and the NSE,GFAP and VIM were all positively expressed in the cells.Conclusion Human BM-MSCs can be cultured and proliferated in vitro,and can differentiate into neuron like cells directionally.