摘要
目的探讨抗坏血酸（ascorbic acid，AA）诱导神经干细胞（neural stem cells，NSCs）向多巴胺能神经元方向分化的作用并初步探索其机制。方法从12.5 d胚胎大鼠中脑获得NSCs，培养扩增后分为对照组和分别加入1、10、50、100、200 μmol/L AA处理组，比较NSCs向多巴胺能神经元分化结果；采用免疫细胞化学对NSCs以及分化后神经元进行鉴定和检测，RT-PCR检测酪氨酸羟化酶（tyrosine hydroxylase，TH）、多巴胺转运子（dopamine transporter，DAT）、SHH、Nurr1、Ptx3 mRNA表达；在AA诱导分化中加入ERK阻断剂 PD98059，以探讨ERK通路在分化中的作用。结果获得大量的神经球并且表达nestin强阳性；AA处理组比对照组表现出较强的多巴胺能分化，其中100 μmol/L AA诱导组表达TH和DAT阳性细胞数量比对照组明显增加（P〈0.05），增加30～50倍；AA诱导增加了Nurr1、TH、DAT、SHH mRNA的表达，ERK抑制剂PD98059能抑制AA诱导后Nurr1、TH、DAT mRNA的表达而不影响SHH mRNA表达（P〈0.05 ）。结论抗坏血酸能诱导神经干细胞分化为多巴胺能神经元，ERK途径可能是AA诱导过程中参与多巴胺能分化的途径之一，Nurr1基因可能参与到ERK途径中或者是其下游基因。
Objective To investigate the ascorbic acid （AA）-induced differentiation of neural stem cells （NSCs） to dopaminergic neurons and explore the mechanism preliminarily. Methods NSCs derived from 12.5 d rat embryo mesencephalon were maintained and amplified. Cells were divided into a control group and AA-induced groups with AA concentration of 1, 10, 50, 100 μmol/L, and 200 μmol/L. The differentiation results of NSCs to dopaminergic neurons in the groups were compared. Immunocytochemistry was carried out to identify NSCs and cell differentiation. The mRNA expression levels of tyrosine hydroxylase （TH）, dopamine transporter （DAT）, SHH, Nurr1, and Ptx3 were tested by RT-PCR. PD98059, an ERK inhibitor, was added during AA-induced differentiation to study the ERK pathway’s role. Results A large number of nestin-positive neurospheres were obtained. Comparing with the control, AA treatment led to a higher degree of dopaminergic differentiation. TH- and DAT-positive cells were increased by about 30-50 folds in the 100 μmol/L AA group as compared to the control group （P〈0.05）. The induction of AA enhanced the mRNA expression of Nurr1, SHH, TH, and DAT. PD98059 could block the mRNA expression of Nurr1, TH, and DAT but not affect the expression of SHH mRNA after AA induction （P〈0.05）. Conclusion AA can induce differentiation of NSCs into dopaminergic neurons. ERK cascade may be one of the pathways involved in AA-induced dopaminergic differentiation. Nurr1 gene may be the downstream gene of ERK pathway or involved in ERK pathway.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第22期2430-2434,共5页
Journal of Third Military Medical University
基金
国家自然科学基金(31100985)
重庆市自然科学基金(CSTC2010BB5096)~~
关键词
神经干细胞
分化
促分裂原活化蛋白激酶
细胞外信号调节激酶通路
信号转导
neural stem cells
differentiation
mitogen-activated protein kinase
extracellular signal-regulated kinase pathway
signal transduction
作者简介
[通信作者]郑敏，E-mail：zradwj625@gmail．com
