胶质源性神经营养因子与白细胞介素1β体外诱导中脑神经干细胞的分化

Differentiation of mesencephalic neural stem cells induced by glial cell line-derived neurotrophic factor and interleukin-1 beta in vitro

背景：在神经干细胞移植治疗帕金森病中,移植细胞的数量及多巴胺能神经元的分化比率是必须解决的问题,而有效的神经干细胞体外增殖与多巴胺能神经元的大量定向诱导分化是解决问题的关键所在。目的：探讨胶质源性神经营养因子与白细胞介素1β体外诱导中脑神经干细胞向多巴胺能神经元的分化。方法：分离妊娠12d小鼠胚胎腹侧中脑,经胰酶消化和机械吹打制成单细胞悬液,离心过滤后机械方法传代培养5~7d的神经球,分组进行诱导分化10~12d,待80%细胞从神经球迁移出来,分化为单细胞时,进行免疫细胞化学鉴定及流式细胞术检测酪氨酸羟化酶阳性细胞率。结果与结论：神经球细胞表达巢蛋白抗原,能分化为神经元特异性烯醇化酶和胶原纤维酸性蛋白阳性细胞。胶质源性神经营养因子与白细胞介素1β在体外能明显提高中脑神经干细胞分化为酪氨酸羟化酶阳性神经元的比例,胶质源性神经营养因子诱导组、白细胞介素1β诱导组和两者联合应用均较空白对照组比例高,尤其是两者联合应用作用更显著,说明胶质源性神经营养因子、白细胞介素1β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,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 neural stem cells into dopaminergic neurons during induction of glial cell line-derived neurotrophic factor（GDNF） and interleukin-1β in vitro.METHODS：Mesencephalon was isolated from mouse embryo ventral part following 12 days of pregnancy,and made into single cell suspension using trypsin digestion and mechanical trituration.Following centrifugation,neurospheres were subcultured by the mechanical method for 5-7 days,and then divided into groups to induce for 10-12 days.When 80% cells migrated from neurospheres differentiated into single cells,immunocytochemical identification and flow cytometry detection were utilized to determine the rate of tyrosine hydroxylase positive cells.RESULTS AND CONCLUSION：Neurospheres expressed nestin antigen,could differentiate into neuron specific enolase and glial fibral acidic protein-positive cells.Glial cell line-derived neurotrophic factor（GDNF） and interleukin-1β could significantly elevate the proportion of the differentiation of mesencephalic neural stem cells into tyrosine hydroxylase-positive neurons.The proportion was higher in the GDNF group,interleukin-1β group and GDNF ＋ interleukin-1β group compared with the blank control group.In particular,the effect of GDNF ＋ interleukin-1β was more significant.Results indicated that GDNF and interleukin-1β can obviously promote the differentiation of mesencephalic neural stem cells into enough number,mature morphology and mature function of dopamine neurons.