【摘要】 目的采用密度梯度离心和差速贴壁法体外分离、培养、纯化及鉴定成年Wistar大鼠骨髓间充质干细胞(MSCs),利用碱性成纤维细胞生长因子(bFGF)和依达拉奉联合诱导MSCs定向分化为神经元样细胞。方法选用1月龄健康雄性Wistar大鼠,采用Percoll分离液密度梯度离心法获取骨髓中的MSCs,通过差速贴壁法反复传代培养、纯化,取第3代细胞采用流式细胞术、免疫细胞化学等方法检测MSCs的纯度,利用bFGF联合依达拉奉诱导MSCs向神经元样细胞分化,并通过扫描电镜、免疫细胞化学等对诱导后的细胞进行鉴定。结果第3代MSCs经免疫细胞化学及流式细胞仪等检测,间充质干细胞特异性的标记物CD44表达阳性,不表达造血干细胞及白细胞的特异性标记物CD34、CD45,MSCs纯度高达95.5%。诱导分化后扫描电镜检测可观察到典型的神经元样细胞结构;免疫细胞化学检测发现,细胞表达神经元特异性烯醇化酶(NSE),不表达胶质纤维酸性蛋白(GFAP),Nestin的表达随着细胞成熟而减弱。结论密度梯度离心和差速贴壁法可获得高纯度的MSCs;依达拉奉能高效地定向诱导MSCs分化为神经元样细胞。更多还原

【Abstract】 Objective To isolate,cultivate and purify rat bone marrow mesenchymal stem cells(MSCs),and induce its directional differentiation into neuron-like cells in vitro by basic fibroblast growth factor(bFGF) and edaravone.Methods One month old healthy male Wistar rats were used in this study,MSCs from the bone marrow were isolated and purificated by Percoll medium density gradient centrifugation,repeated subculture and differential adherence methods.After 3 passages,the surface markers of MSCs were detected by immunocytochemistry and flow cytometry.Cell differentiation of MSCs induced by bFGF and edaravone was explored by immunocytochemistry and scanning electron microscopy.Results The cell purity of MSCs was up to 95%.The purified MSCs expressed CD44,but did not express CD34 and CD45.After directional differentiation cultivation,the cells showed the typical neuron appearance cell under the scanning electron microscope and expressed neuron-specific enolase but did not express glial fibrillary acidic protein.Nestin expression in the cells was gradually weakened.Conclusion The results suggest that density gradient centrifugation and differential adhesion methods may be one of the suitable methods of purification of the MSCs,and edaravone can direct and efficiently induce MSCs to differentiate into neuron-like cells.更多还原