脐带间充质干细胞源性多巴胺能神经元中脑源性神经生长因子的表达研究

Study on the expression of brain-derived neurotrophic factor in human umbilical cord mesenchymal stem cell-derived dopaminergic neurons

目的将人脐带间充质干细胞(HUCMSCs)诱导分化为多巴胺能神经元并观察其中脑源性神经营养因子(BDNF)的表达。方法将P3代脐带MSCs接种24孔细胞培养板,培养24 h后更换神经诱导液,诱导9 d后加入BDNF。终止诱导后应用免疫荧光染色检测细胞中酪氨酸羟化酶(TH)、β-微管蛋白Ⅲ、神经元核抗原(NeuN)和BDNF的表达。结果 HUCMSCs经特定细胞因子诱导后可分化形成成熟的神经元,分化比例约为70%;其中定向分化形成TH阳性细胞的比例为(17.65±1.91)%;BDNF表达比例为(14.15±3.04)%。结论 HUCMSCs经诱导后可向多巴胺能神经元分化,且分化后的细胞可表达BDNF。

Objective To induce human umbilical cord mesenchymal stem cells(HUMSCs) to differentiate into dopaminergic neurons,and to detect the expression of brain-derived neurotrophic factor(BDNF) in them.Methods The P3 HUCMSCs were seeded in the density of 1 × 104/ well in 24-well plates for 24 h,then the medium was replaced by induction medium,containing DMEM / F12,50 μg / ml vitamin c,250 ng / ml sonic hedgehog(SHH),100 ng / ml fibroblast growth factory 8(FGF8),50 ng / ml basic FGF(bFGF),(100 ×) N2.Nine days later,BDNF(50 ng / ml) was added to the medium.The markers and neurotrophic factor of neurons,including β-tubulin Ⅲ,neuronal nuclear antigen(NeuN),tyrosine hydroxylase(TH) and BDNF,were detected by immunocytochemistry.Results Before being induced,few of MSCs expressed β-tubulin Ⅲ(21.24 ± 0.97) % and no MSCs expressed NeuN,TH and BDNF.After being induced,MSCs derived from human umbilical cord differentiated into dopaminergic neurons.Cell morphology changed obviously,and the expression of β-tubulin Ⅲ,NeuN,TH and BDNF increased.The ratio of positive cells of β-tubulin Ⅲ,NeuN,TH and BDNF was(55.5 ± 11.67) %,70.0 ± 7.52) %,(17.65 ± 1.91) % and(14.15 ± 3.04) %,respectively.Conclusions HUCMSCs can be induced and differentiated into dopaminergic neurons which can express BDNF in vitro.