不同浓度GDNF诱导神经干细胞向多巴胺能神经元分化研究

Research on the induction of differentiation of neural stem cells with dopaminergic neurons by different concentrations of glial cell line-derived neurotrophic factors

目的观察不同浓度胶质细胞源性神经营养因子(GDNF)对神经干细胞(NSCs)向多巴胺(DA)能神经元分化的影响。方法取新生鼠脑组织体外分离培养NSCs,第2代NSCs诱导培养基中分别加入0、5、10、20 ng/mL GDNF进行诱导。用逆转录-聚合酶链反应检测诱导后细胞酪氨酸羟化酶(TH)mRNA的表达,免疫细胞化学染色鉴定NSCs,检测NSCs向DA能神经元分化率。结果各诱导组均表达TH mRNA。神经球具有自我更新和表达巢蛋白的能力,诱导分化后的细胞能表达神经元、星形胶质细胞及少突胶质细胞特异性抗原。与空白对照组比较,GDNF诱导组TH阳性细胞率均明显升高(P<0.05);且10、20ng/mL GDNF诱导组升高幅度明显高于5 ng/mL GDNF诱导组(P<0.05),10、20 ng/mL GDNF诱导组间比较差异无统计学意义(P>0.05)。结论从新生鼠脑组织分离出NSCs。不同浓度的GDNF均能促进NSCs向DA能神经元分化,GDNF浓度从10ng/mL提高到20 ng/mL时TH阳性细胞率无明显变化。

Objective To investigate the effects of glial cell line-derived neurotrophic factor with different concentrations on the differentiation of neural stem cells（NSCs） into dopamine（DA） neurons in vitro.Methods The primary NSCs were isolated from the whole brains of neonatal rats.0,5,10,20 ng/mL GDNFs were added into the second generation of NSCs to induce DA neurons for 10 days.Tyrosine hydroxylase（TH） mRNA was detected by RT-PCR;NSCs and DA neurons differentiation from NSCs were detected by immunocytochemiscal stain assay.Results All groups could express TH mRNA.The neurospheres neurospheres had the capacity of self-renewing,expression of nidogen,and could differentiate into multi-directions.Specific antigens of neurons,astrocytesand oligodendrocytes were also expressed.Compared with blank control group,GDNF increased the proportion of TH-positive cells（P0.05）,10,20 ng/mL GDNF were significantly higher than that of 5 ng/mL group（P0.05）,while no significant difference was found between 10,20 ng/mL GDNF groups（P0.05）.Conclusion The NSCs were isolated from the brains of neonatal rats.GDNF could promote the differentiation of NSCs into DA neurons,and TH positive cell rate changed minimally when GDNF was increased from 10 to 20 ng/mL.