Objective :To explore the feasibility that human umbilical cord mesenchymal stem cells (hUCMSCs) differentiated into motoneurons induced by retinoic acid (RA) and sonic hedgehog (Shh), thereafter, to provide experimental basis for clinical translation. Methods-, The hUCMSCs were isolated and purified by double enzymatic digestion and the surface antigens of these cells were detected by flow cytometry. According to different inducer. the experimentation was divided into four groups; control (no inducer), RA group (0. 5 μg/ml RA), Shh group (300 ng/ml Shh) and RA+Shh group (0. 5 μg/ ml RA+300 ng/ml Shh). After hUCMSCs being induced for 25 days, the morphological change was observed, and the expressions of Nestin, MAP-2, Hb9 and GFAP were detected by immunocytochemical method. Additionally, the immunocy-tochemical analysis for these four proteins was also performed on day 5, 10, 15, and 20 after induction in RA+Shh group. Results-. hUCMSCs were positive to CD29 and CD44, and negative to CD31 and CD34. In the control and Shh group, morphological change of cells was not found, and the proportion of positive cells of Nestin and MAP-2 was low. Huwever, the cells induced by RA only or combination of RA+Shh presented typical neuronal-like appearance exhibited as cytoplasm contracted towards the nucleus and processes were formed and connected like network. The proportion of positive cells of Nestin and MAP-2 was significantly higher than that in the other groups. In four groups, positive to Hb9 was only found in RA+ Shh group. And, the GFAP was weak expressed without significant difference in four groups. The combination of RA and Shh caused detectable Nestin on day 5, MAP-2 on day 10, Hb9 on day 15. On day 20, the Nestin expression was decreased, and MAP-2 and Hb9 reached their highest levels on day 25. Conclusion-. RA could greatly promote the differentiation of hUCMSCs into neurons, while solitary Shh have no effect on it. However, hUCMSCs can be induced into the motoneuronsrnwith combination of RA and Shh.%目的:探讨单独和联合应用维甲酸(RA)与音猬因子(Shh)对人脐带间充质干细胞(hUCMSCs)向运动神经元分化的影响,为其进一步临床应用提供理论基础.方法:用双酶消化法提取纯化hUCMSCs,取传至第3代的hUCMSCs,检测表面抗原CD29、CD31、CD34、CD44的表达情况.根据培养液中加入不同诱导剂分为空白对照组,RA组(0.5μg/ml RA),Shh组(300 ng/ml Shh),RA+Shh组(0.5μg/ml RA和300 ng/ml Shh).诱导细胞培养后第25天,观察细胞形态的变化；采用免疫细胞化学检测Nestin、MAP-2、Hb9和GFAP的表达,比较各组阳性率.用免疫印迹检测RA+Shh组诱导后第5、10、15、20、25天Nestin、MAP-2、Hb9的表达情况.结果:流式细胞仪检测结果显示,CD29、CD44高表达,CD31、CD34阴性.空白对照组和Shh组细胞诱导后形态变化不明显,Nestin和MAP-2阳性表达率均很低,两者之间无差别.RA组和RA+Shh组细胞诱导第25天后,细胞胞质以胞核为中心收缩,部分胞质收缩形成细胞的突起,突起之间有网状连接,具有典型的神经元样形态；Nestin和MAP-2的阳性表达率比其他2组增多.4组中只有RA+Shh组细胞诱导后Hb9表达阳性,阳性率为33.62％,其他各组均无Hb9表达.各组GFAP表达阳性率均较低,无差异.免疫印迹检测结果显示,RA+Shh组细胞从第5天起,可见Nestin的表达；第10天少量的MAP-2表达；第15天Hb9开始出现；第20天Nestin下降；第25天MAP-2和Hb9达到本次检测的高峰.结论:RA能促进hUCMSCs向神经元分化,单独使用Shh并没有明显效果,而RA联合Shh能促进hUCMSCs向运动神经元分化.
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