维甲酸联合Purmorphamine诱导人脐带间充质干细胞向运动神经元分化的研究  

Differentiation of human umbilical cord mesenchymal stem cells into motoneurons induced by retinoic acid combined with purmorphamine

作　　者：刘学元[1] 李德华[1] 单伟[1] 姜东[1] 

机构地区：[1]辽宁医学院解剖学教研室,锦州121000

出　　处：《神经解剖学杂志》2011年第6期647-652,共6页Chinese Journal of Neuroanatomy

基　　金：辽宁省自然科学基金项目(20102138);辽宁医学院青年科技启动基金资助项目(Y2010Z004)

摘　　要：目的:探讨单独和联合应用维甲酸(retinoic acid,RA)与purmorphamine(2,6,9-三取代嘌呤化合物,PM)对人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)向运动神经元分化的影响,为其进一步临床应用提供实验依据。方法:用双酶消化法提取纯化hUCMSCs,取传至第3代的hUCMSCs,流式细胞仪检测表面抗原CD29、CD31、CD34、CD44的表达情况。根据加入不同诱导剂分4组:空白对照组;RA组(加入0.5μg/ml RA);PM组(加入1μg/ml PM);RA+PM组(加入0.5μg/ml RA和1μg/ml PM)。细胞诱导后第20d,观察细胞形态的变化;采用免疫细胞化学方法检测Nestin、β-tubulin-III、Hb9、ChAT的表达情况,比较各组细胞阳性表达的比例。RA+PM组细胞诱导5、10、15、20、25 d后收集细胞,检测细胞内Ach的含量。结果:流式细胞仪检测结果显示,CD29、CD44高表达,CD31阴性,CD34不表达。空白对照组和PM组细胞诱导后形态变化不明显,Nestin和β-tubulin-III阳性表达率均很低,两者之间无差别。RA组和RA+PM组的诱导后细胞具有典型的神经元样形态;且Nestin和β-tubulin-III阳性表达率比其他两组高,有显著性差异(P<0.01)。4组中只有RA+PM组细胞诱导后出现Hb9表达阳性,其他各组均无Hb9表达。RA+PM组ChAT高表达,RA组仅少量表达。从第15 d起,RA+PM组细胞可检测到Ach,其含量随着诱导时间的延长逐渐增加。结论:RA能促进hUCMSCs向神经元分化,单独使用PM并没有明显效果,而RA联合PM能显著促进hUCMSCs向运动神经元分化。Objective： To study the effects of retinoic acid（RA） alone or combined with purmorphamine（PM） on the differentiation of the human umbilical cord mesenchymal stem cells（hUCMSCs） into the motoneurons,and to provide the experimental data for further clinical practice.Methods： Purified hUCMSCs were extracted with the double enzyme digestion method.The expressions of the surface antigen CD29,CD31,CD34,CD44 were detected on the 3rd generation of hUCMSCs.Four groups were classified according to the different components of RA and PM in medium： Control group;RA group（0.5 μg/ml RA）;PM group（1 μg/ml PM）;RA＋PM group（0.5 μg/ml RA and 1 μg/ml PM）.On the 20th day after being induced,the morphologic changes of the cells were observed.The expressions of Nestin,β-tubulin-III,Hb9 and ChAT were detected via immunocytochemical methods,and the expressional rates of positive cells in each group were compared.In RA＋PM group,the cells were collected on the 5 th,10 th,15 th,20 th,25 th day after being induced and the contents of Ach were detected.Results： The flow cytometry testing results showed that CD29,CD44 was with high expression,CD31 negative expression,CD34 with no expression.In the control group and PM group,there were no obvious changes in morphology after being induced,and the expressional rates of Nestin and β-tubulin-III positive cells were both very low,with no difference between them.In RA group and RA＋PM group,the cells took on a typical neuronal morphology,and the expressional rates of Nestin and β-tubulin-III positive cells were both higher than those in the other two groups,with significant differences（P〈0.01）.In all four groups,the expression of Hb9 was positive only in RA＋PM group and with no expression in the other three groups.There were high expression of ChAT in RA＋PM group and low in RA group.From the 15th day,Ach can be detected in RA＋PM group,and its content increased with the induction time.Conclusion： RA could promote the differentiation of hUCMSCs in

关 键 词：人脐带间充质干细胞 运动神经元 维甲酸 purmorphamine 诱导 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]