常氧及低氧条件下胶质源性神经营养因子体外诱导中脑神经干细胞向多巴胺能神经元的分化

Glial-derived neurotrophic factor induces the in vitro differentiation of mesencephalic neural stem cells into dopaminergic neurons under normoxic and hypoxic conditions

背景:神经干细胞的定向诱导分化和扩增受细胞自身基因和外来信号的调控。目的:观察中脑源性神经干细胞在常氧、低氧和胶质源性神经营养因子诱导下向多巴胺能神经元的分化情况。方法:无菌条件下分离E12小鼠胚胎腹侧中脑组织,胰酶消化和机械吹打制成单细胞悬液,在无血清培养基中培养扩增;Nestin免疫细胞化学染色方法鉴定神经干细胞。在有血清培养基中对纯化神经干细胞自然分化;神经元特异性烯醇化酶和胶质纤维酸性蛋白免疫细胞化学染色方法分别鉴定神经元和星形胶质细胞。建立常氧和低氧环境,设置常氧组、常氧+胶质源性神经营养因子组、低氧组、低氧+胶质源性神经营养因子组,按实验分组在有血清条件下诱导分化。结果与结论:在低氧条件下,中脑神经干细胞向多巴胺能神经元分化均高于常氧组;尤其是低氧环境和胶质源性神经营养因子诱导下向多巴胺能神经元分化比例更高,表型更成熟。说明低氧环境下胶质源性神经营养因子可明显促进中脑神经干细胞分化为数量足够、形态及功能成熟的多巴胺能神经元。

BACKGROUND：Directed differentiation and proliferation of nerve stem cells are regulated by their own genes and external signals.OBJECTIVE：To observe the differentiation of mesencephalic neural stem cells into dopaminergic neurons under normoxic and hypoxic conditions induced by glial-derived neurotrophic factor（GDNF）.METHODS：Under aseptic conditions,we isolated mesencephalic tissues from E12 mouse embryos.Single cell suspension was prepared by pancreas enzyme digestion and mechanical pipetting,and were cultured and proliferated in serum-free medium.Nestin immunocytochemistry staining was used to identify nerve stem cells.Purified nerve stem cells differentiated in serum medium.Neuron-specific enolase and glial fibrillary acidic protein immunocytochemistry staining methods were performed to identify neurons and astrocytes,respectively.All the cells were divided into normoxia group,normoxia＋GDNF group,hypoxia group,hypoxia＋GDNF group,and then cultured in serum medium.RESULTS AND CONCLUSION：Under the hypoxia condition,mesencephalic neural stem cells had a higher differentiated rate than that in the normoxia group.The number of differentiated cells in the hypoxia＋GDNF group was higher than that of the other groups,and the differentiated cells had a mature phenotype.It is indicated that under the hypoxic condition,GDNF can extremely promote the differentiation of nerve stem cells into dopaminergic neurons with good morphology and mature function.