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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RealtimePCR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。
AIM: To investigate the effects and mechanisms of human amniotic epithelial cell conditioned medium (ACM) and SHH and FGF8-induced differentiation of human umbilical cord blood mesenchymal stem cells (CB-MSCs) into dopamine (DA) neuron-like cells. Methods: CB-MSCs were purified by sedimentation of erythrocytes, density gradient centrifugation and adherent screening. ACM and SHH were added into the culture medium. The FGF8 was divided into four groups: CON, ACM, SHH + FGF8 and ACM + SHH + FGF8 for 48h. Immunocytochemical staining Identification of differentiated cells DA neuronal phenotype. The content of DA in cell culture supernatant was determined by HPLC. The expression of En-1, Foxa2, Lmx1b, Gli-1, Pitx3, Nurr1 and Ngn2 in DA neurons was detected by Realtime PCR. Results: After induction with inducer for 48h, the expression of TH and DAT in each induction group were higher than those in control group. The early transcription factors En1 and Foxa2 were expressed in all the groups, whereas Pitx3 and Lmx1b were expressed in each induction group, and Lmx1b was the highest expression in ACM + SHH + FGF8 group. Pitx3 was expressed in all induction groups difference. Nurr1 and Ngn2 expressed strongly in SHH + FGF8 group. CONCLUSION: ACM, SHH and FGF8 can induce the differentiation of umbilical cord blood mesenchymal stem cells into DA neuron-like cells. The mechanism is related to the expression of En-1, Foxa2, Lmx1b and Gli-1 in dopaminergic neurons , Pitx3, Nurr1, and Ngn2.