诱导人脐血间充质干细胞分化为多巴胺能神经元样细胞的研究

目的：探讨人羊膜上皮细胞条件培养液(ACM)及SHH，FGF8诱导人脐血间充质干细胞（CB-MSCs）分化为多巴胺( dopamine,DA )能神经元样细胞的作用及机制。方法：利用沉降红细胞、密度梯度离心和贴壁筛选法纯化CB-MSCs，加入ACM及SHH，FGF8分为CON，ACM，SHH+FGF8，ACM+SHH+FGF8四组诱导48h后，免疫细胞化学染色鉴定分化细胞的DA神经元表型。应用高压液相色谱技术测定细胞培养上清中DA含量，并应用Realtime PCR方法观察DA能神经元发育过程中的相关基因En-1，Foxa2，Lmx1b，Gli-1，Pitx3，Nurr1以及Ngn2的表达。结果：加诱导剂诱导48h后，各诱导组的TH和DAT的表达量均比对照组高。早期的转录因子En1，Foxa2 在各组中均有表达；而Pitx3，Lmx1b表达在各诱导组中；Lmx1b在ACM+SHH+FGF8组中表达最高；Pitx3在各诱导组中均有表达且无明显差异。Nurr1，Ngn2在SHH+FGF8组中表达很强。结论：ACM及SHH，FGF8可诱导脐血间充质干细胞分化为DA能神经元样细胞，其作用机制与多巴胺能神经元发育过程中的各种基因En-1，Foxa2，Lmx1b，Gli-1，Pitx3，Nurr1以及Ngn2相关。

The Study of CB-MSCs Differentiating to Become Dopaminergic Neuron-like Cells

Abstract : Objective :To detect the dopaminergic neural cells differentiated pathway, the related gene in dopaminergic neural cells development process were investigated. The role of Human amnion epithelial conditional media (ACM) and SHH, FGF8 was focused on in cultivation process of human umbilical cord blood mesenchymal stem cells (CB-MSCs). Method: Preparation of the mononuclear cell fraction was performed by the Ficoll gradient technique according to the manufacturer’s instructions . ACM and SHH, FGF8 were combined to induce CB-MSCs, and there were four groups CON, ACM, SHH+FGF8, ACM+SHH+FGF8. After 48 hours of co-cultivation , Immunophenotypes of CB-MSCs were observed by immunocytochemistry and the related gene in dopaminergic neural cells development process En-1、Foxa2、Lmx1b、Gli-1、Pitx3，Nurr1 and Ngn2 were detected by RT-PCR. Results: After 48 hours of co-cultivation ，the positive cells numer of TH and DAT were higher in induction groups than in control group. By RT-PCR, En-1，Foxa2，Pitx3, Lmx1b were higher in ACM, SHH+FGF8, ACM+SHH+FGF8 groups than in the control group(p<0.01). Nurr1,Ngn2 were strongly higher in SHH+FGF8. Conclusion:The related gene in dopaminergic neural cells development process，En-1、Foxa2、Lmx1b、Gli-1、Pitx3，Nurr1 and Ngn2 play a role in the differentiated pathway form CB-MSCs to dopaminergic neural cells.