神经干细胞定向分化为多巴胺能神经元过程中Mash1及Hes1基因的表达

The expression of Mash1 and Hes1 during induce neural stem cells differentiating into dopaminergic neurons

目的建立神经干细胞(NSCs)分化为多巴胺能神经元的体外模型,并进一步研究此分化过程中Mash1及Hes1基因的表达变化。方法无菌条件下取孕14-16d胎鼠端脑进行NSCs体外培养,将NSCs进行Nestin免疫荧光检测,对照组和实验组(GDNF+IL-1α联合组)诱导2周后进行β-tubulinⅢ和TH免疫荧光染色,计算各组阳性细胞率,荧光定量PCR技术检测P4代NSCs分化1周和2周时Mash1及Hes1基因的表达变化。结果免疫荧光检测结果显示,NSCs的Nestin阳性细胞率为(93.04±3.55)%,诱导分化2周后对照组、实验组β-tubulinⅢ阳性细胞率分别为(12.65±1.58)%和(33.95±2.97)%,两组间比较差异存在统计学意义(P<0.05)。对照组、实验组TH阳性细胞率分别为(1.46±0.41)%、(15.51±1.94)%,差异存在统计学意义(P<0.05)。荧光定量PCR结果显示,NSCs诱导分化后,Mash1基因表达较未分化状态时升高,各组间比较差异存在统计学意义,实验组高于对照组(P<0.05),分化2周高于1周(P<0.01);Hes1基因于分化后表达降低,对照组与对实验组之间以及分化1周与2周之间比较差异没有统计学意义(P>0.05)。结论 GDNF和IL-1α联合作用能够诱导NSCs定向分化为多巴胺能神经元;Mash1与Hes1基因可能参与NSCs分化为多巴胺能神经元的过程。

Objective To build the in vitro modeld of NSCs differentiate into dopaminegic neurons,and further study the expression of Mash1 and Hes1 in this process.Methods Separated the telencephalon of rat embryos in sterile condition and cultured in vitro,take NSCs stained by Nestin immunofluorescence assay,divided into con=group and exp-group（GDNF＋IL-1α） and induced into dopaminegic neurons,after 2 weeks stained by β-tubulinⅢ and TH,and counted the rate of positive cells of each group.q-PCR checked the expression of Mash1 and Hes1 in NSCs and during differentiated into dopaminegic neurons.Results IF showed that the rate of Nestin positive cells was（93.04±3.55）%,There were no statistical difference between the two comparison（P〈0.05）.After differentiated 2 weeks,the rate of β-tubulinⅢ positive cells were：P4,con-group [（12.65±1.58）%],exp-group[（33.95±2.97）%],respectively,There were statistical difference between the two comparison（P〈0.05）; the rate of TH positive cell were：P4,con-group [（1.46±0.41）% ],exp-group [（15.51±1.94）% ],There were statistical difference between the two comparison（P〈0.05）.q-PCR result showed that after differentiation in P4 NSCs,Mash1 expression was higher than NSCs,there were statistical difference between each group,exp-group higher than con-group（P〈0.05）,2 weeks higher than 1 week（P〈0.01）; Hes1 expression was lower than NSCs,there were statistical difference compared with each group（P〈0.05）.Conclusion The combined effect of GDNF and IL-1 was able to induce NSCs differentiating into dopaminergic neurons; Mash1 and Hes1 gene maybe involve in NSCs differentiated into dopaminergic neurons.