低氧环境下胶质源性神经营养因子体外诱导中脑神经干细胞的分化

In vitro differentiation of mesencephalic neural stem cells induced by glial-derived neurotrophic factor under hypoxia

背景：有效的神经干细胞体外增殖与多巴胺能神经元的定向诱导分化是神经干细胞移植治疗帕金森病的关键所在.目的：观察低氧条件下胶质源性神经营养因子体外诱导中脑源性神经干细胞向多巴胺能神经元的分化.方法：体外分离培养孕12 d胚鼠腹侧中脑组织,制成单细胞悬液,在含碱性成纤维细胞生长因子和B27的无血清培养基中培养并传代,分别置于常氧（体积分数21%O2）或低氧（体积分数3%O2）环境下增殖5~7 d后,接种于含体积分数10%胎牛血清的DMEM/F12培养基,或含体积分数10%胎牛血清的DMEM/F12＋1 μg/L胶质源性神经营养因子.结果与结论：低氧环境下分化10~12 d,中脑神经干细胞向多巴胺能神经元分化均高于常氧组,在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： During neural stem cell transplantation in the treatment of Parkinson’s disease, the number of transplanted cells and differentiation ratio of dopaminergic neurons must be resolved. Effective in vitro proliferation of neural stem cells and large amount of directed differentiation of dopaminergic neurons are the key to solve above-mentioned problems. OBJECTIVE： To investigate the differentiation of mesencephalic neural stem cells into dopaminergic neurons during the hypoxia induced by glial cell line-derived neurotrophic factor （GDNF） in vitro. METHODS： Ventral midbrain tissue isolated from embryonic mice of pregnant 12 days was made into single cell suspension and cultured in non-serum medium containing basic fibroblast growth factor （bFGF） and B27, and then proliferated under normoxia or hypoxia for 5-7 days followed by incubation in DMEM/F12 medium containing 10% fetal bovine serum or 10% fetal bovine serum ＋ 1 g/L GDNF. RESULTS AND CONCLUSION： Under the hypoxia environment, the number of dopaminergic neurons differentiated from mesencephalic neural stem cells especially induced by GDNF was higher than that under normoxia. It indicated that under the hypoxia environment, GDNF can induce mesencephalic neural stem cells differentiating into dopaminergic neurons with enough quantity, mature shape and function.