视网膜细胞联合bFGF体外诱导大鼠骨髓间充质干细胞向神经样细胞的分化

目的 探讨大鼠骨髓间充质干细胞（BMSC）在体外分化为神经样细胞的有效途径,从而解决体外诱导分化效率低及存活状况不佳等问题.方法 采用密度梯度离心法和贴壁筛选法分离BMSC,免疫组化检测CD31、CD44、CD45、CD105表达并对细胞进行鉴定.按照诱导方式的不同分为3组：视网膜细胞＋碱性成纤维细胞生长因子（bFGF）组、bFGF组、不加诱导液组,分别诱导大鼠BMSC向神经样细胞分化.于诱导后第3、第7、第14天分别进行细胞形态学观察;并采用免疫细胞化学法检测神经微管蛋白-βⅢ（Tui1）、神经元特异性烯醇化酶（NSE）和胶质纤维酸性蛋白（GFAP）的表达,分析阳性细胞率：采用MTT法检测诱导后BMSC增殖状况,比较细胞存活率.相同时间组间比较用两独立样本t检验,组内不同时间比较用单因素方差分析.结果 形态学观察：视网膜细胞＋hFGF组诱导BMSC 12 h后出现形态变化,逐步形成典型的神经样细胞：bFGF阳性对照组也可见形成突起结构,但无典型的神经样细胞形态.免疫组化检测：处理组诱导3 d后即可检测到阳性细胞,随着诱导时间的延长.Tui1、NSE和GFAP阳性细胞率增加,与阳性对照组比较差异有统计学意义（P〈0.01）;阴性对照组未发现阳性细胞.存活率：各组BMSC存活率随着时间延长逐渐下降,但不同诱导方法对BMSC存活无显著影响.结论 模拟视网膜微环境配合bFCF能够诱导大鼠BMSC分化为神经样细胞并继续存活.

Differentiation of rat bone marrow mesenchymal stem cells into nerve-like cells through co-culture with retinal cells and application of basic fibroblast growth factor in vitro

Objective To investigate an effective way to differentiate bone marrow mesenchymal stem cells (BMSC) into nerve-like cells and address the low percentage of differentiation and survival of BMSC induced in vitro. Methods BMSC was separated using density gradient centrifugation and adherence screening methods. The surface antigen of BMSC was identified by immunohistochemistry methods using CD31, CD44, CD45, CD105 monoclonal antibody. The experimental groups were divided as follows: a retinal cell plus basic fibroblast growth factor (bFGF) group, bFGF group, and a negative control group. The expression of Neuronal Class Ⅲβ -Tubulin (Tuj1), neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) of induced BMSC was observed on day 3, 7 and 14 by immunocytochemistry. Animation of induced BMSC was observed by the MTT method. Results The morphologic shape of induced BMSC changed after 12 hours and gradually became typical nerve-like cells. Tuj1, NSE and GFAP positive cells could be found after three days by immunocytochemistry. The percentage of these positive cells increased in the total population with induced time. The proliferative ability of the induced BMSC had not been affected markedly. Conclusion An analogical retinal microenvoirenment and bFGF can induce BMSC to differentiate into nerve-like cells and survive in vitro.