神经营养因子对体外培养中脑多巴胺能神经元存活和分化的影响

目的观察不同神经营养因子对体外培养中脑多巴胺能神经元(DN)存活和分化的作用。方法选取14d孕鼠,无菌条件下取出胎鼠,采用酶消化法培养中脑DN神经元,在培养过程中,分别加入不同浓度的胶质细胞源性神经营养因子(GDNF)、神经营养因子3(NT3)、脑源性神经营养因子(BDNF)和神经生长因子(NGF),通过细胞形态学和免疫荧光方法进行细胞纯度鉴定,观察不同作用条件下TH阳性细胞率确定细胞存活。结果以10~60ng/L的GDNF或BDNF持续培养10d,中脑多巴胺能神经元的存活率明显高于NGF和NT3作用组,浓度为20ng/m l的GDNF作用最强,能够维持60%的DN神经元存活。此外BDNF和GDNF能够增加DN神经元的数目,但未发现明显的剂量依赖效应,当GDNF与BDNF联合应用时,未见DN神经元的保护作用增强。结论GDNF和BDNF对原代培养的多巴胺能神经元存活具有较强的促进作用,并能诱导神经前体细胞分化为DN神经元。

Effect of neurotrophins on cultured dopaminergic neurons' survival and differentiation in vitro

Objective To explore the effect of different neurotrophic factors on primary cultured dopaminergic(DN) neurons' survival.Methods The DN neurons were obtained from the rostral mesencephalic tegmentum of Wistar rat embryos(E14) by using trypsin and collagenase method.The cells were treated with GDNF,BDNF,NT3 and NGF of various concentrations(0,10ng/ml,20ng/ml,40ng/ml and 60ng/ml) respectively.The cell morphology and immunofluorescence staining of TH antibody was used to labelled DN neurons to observe the neurons survival.Results In GDNF and BDNF group,the rate of DN neurons on 10d after culture significantly increased than in NT3 and NGF group.60% of DN neurons survival was seen with 20ng/ml of GDNF.Meanwhile,increased DN neurons was observed in GDNF and BDNF group on 14d than 10d after culture.There was no dose-dependent effect was found in 20-60ng/ml treatment and no significant difference in DN neurons' survival in combined GDNF and BDNF than alone treatment.Conclusion GDNF and BDNF provide protection on primary cultured DN neurons and induce differentiation of neural precursors into dopaminergic neurons.