成年骨髓间质干细胞体外诱导分化成神经细胞研究

目的：探索成年骨髓间质干细胞（ABMMSC）诱导分化为神经细胞（神经元和神经胶质细胞）的可行性，为ABMMSC在神经科学领域内的应用提供 参考。方法：以成年犬ABMMSC为实验对象，利用碱性成纤维细胞生长因子（bFGF）、表皮生长因子（FGF）、维甲酸（RA）、脑源性神经营养因子（BDNF）、胶质细胞系源性神经营养因子（GDNF）等作为增殖及分化诱导因子，采用两步法进行增殖培养，分化诱导；免疫细胞化学法进行细胞性质鉴定。结果：加入bFGF、EGF后增殖培养48h，换液、去除非粘附细胞，再增殖培养72h ,可见细胞分裂相（成纤维细胞样细胞）和簇样克隆形成（中小型细胞）。加入RA、BDNF、GDNF诱导3d，部分细胞有神经元特异性烯醇酶（NSE）、胶质纤维酸性蛋白（GFAP）成分表达；第10d可见有神经元、神经胶质形态样细胞形成。经细胞成分（NSE、GFAP）鉴定证实为神经元、神经胶质细胞。结论：ABMSC在体外培养条件下，经过bFGF、EGF、RA、BDNF、GDNF等因子的“程序性”作用，可以向神经元、神经胶质前体细胞及其终末细胞方向分化。

Adult bone marrow mesenchymal stem cells differentiate into neural cells in vitro

Objective To Explore the feasibility of differentiation into neural cells from adult bone marrow mesenchymal stem cells (ABMMSC)in vitro, and to offer reference for the applying of ABMMSC in the field of neuroscience. Methods The ABMMSC was acquired from adult dog. Basic fibroblast growth factor (bFGF) , epidermal growth factor (EGF) , all - trans - retinoic acid (RA) , brain derived neurotrophic factor (BDNF), glial cell line derived neurotrophic factor (GDNF) served as proliferation or differentiation factors. Two - step methods of expansion and differentiation culture were used in this experiment. Immunohistochemistry was used to identify the cell types. Results Three types of cells, large flat fibroblast - like cells, middling round/ oval cells and small round cells, still adhered on the bottom surface of culture plate after the medium changed and the non - adherent cells were discarded, when bFGF and EGF had been added into the culture medium and the cells had been cultured for 48 hours. At 72 h, cleavage phase of large flat fibroblast - like cells and cluster - like clones of middle/small type cells appeared. On the 3rd day after adding RA, BDNF and GDNF into the culture medium, part of the BMSC derived cells started to express neuron - specific enolase ( NSE) or glial fibrillary acidic protein (GFAP). The neural cell modality -like cells appeared on 10th day after adding inducing factors into the culture medium, and which was proved by the tests of NSE or GFAP expressing. Conclusion The ABMMSC can differentiate into neural cells in vitro, being induced programmatically by such factors as bFGF, EGF, RA, BDNF and GDNF through two -step methods of proliferation and differentiation culture.