诱导人骨髓间充质干细胞向运动神经元分化的研究  

Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Motor Neurons in Vitro

作　　者：孔卫国[1] 吴晓牧[1] 张昆南[1] 胡国柱[2] 丁伟荣[2] 何丹[2] 

机构地区：[1]江西省人民医院神经内科,南昌330006 [2]江西省人民医院临床医学研究所,南昌330006

出　　处：《江西医学院学报》2009年第9期1-4,F0002,共5页Acta Academiae Medicinae Jiangxi

基　　金：国家科技支撑计划课题(2008BAI68B03)

摘　　要：目的探讨人骨髓间充质干细胞(human bone marrow mesenchymal stemcells,hMSCs)在体外诱导向运动神经元分化的潜能。方法用密度梯度离心法分离hMSCs,在含有10%FBS的DMEM/F12培养基中的培养扩增。将传代后的第1代hMSCs分为诱导组和对照组,诱导组的细胞在完全培养基中加入终浓度为10μg/L碱性成纤维细胞生长因子(bFGF)预处理24h后,用0.5μg/mL维甲酸(RA)和200ng/mL音速波状蛋白(shh)联合诱导10d。对照组不加上述诱导剂。光镜下观察其分化过程中hMSCs的形态变化,免疫组化检测诱导前后细胞是否表达神经干细胞特异性标志蛋白,用流式细胞仪检测是否产生运动神经元及在总的细胞数种所占比率,并用流式软件分析结果。结果诱导组hMSCs经诱导后能分化为具有典型神经元形态的细胞,部分细胞表达抗人神经巢蛋白(Nestin),表达率达到(25.0±4.8)%;流式检测诱导后运动神经元特异性标志蛋白HB9表达,表达率达到(50.0±5.3)%。对照组细胞形态无改变,无Nestin表达,HB9表达小于5%。结论hMSCs在体外条件下能诱导分化为运动神经元样细胞。Objective To explore the differentiation potentiality of human bone marrow mesenchymal stem cells （hMSCs） into motor neurons in vitro.Methods The hMSCs were isolated from fresh bone marrow by a method of density gradient centrifugation,then amplified and cultivated in a total medium of DMEM/F12 containing 10% FBS.hMSCs were divided into two groups in the first passage.In the induction group,hMSCs were pretreated with a total mediem consisting of 10 μg/L basic fibroblast growth factor （bFGF） for 24 h and then induced with 0.5 μg/mL ratinoic acid （RA） and 200 ng/mL sonic hedgehog （shh） for 10 days.On the other hand,no inductive agents were added in the control group.After the induction,hMSCs were observed using phase-contrast microscopy.The special mark of neural stem cells were detected by immunocytochemistry and the percentages of positive cells were calculated.The tag of motor neurons were detected by flow cytometry and analyzed by software.Results Under induction conditions,some hMSCs resumed typical neuronal morphology,and the percentages of Nestin,HB9 positive cells were （25.0±4.8）%、（50.0±5.3）% respectively.But in the control group,hMSCs remained unchange morphologically and none of them expressed nestin,and the expression of HB9 was less than 5%.Conclusions hMSCs could be induced to differentiate into motor neurons in vitro.

关 键 词：骨髓间充质干细胞 运动神经元 诱导 分化 

分 类 号：R349.5[医药卫生—基础医学]