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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 establish an in vitro model of neural stem cells (NSCs) differentiating into dopaminergic neurons and further investigate the expression of Mash1 and Hes1 genes in this differentiation process. Methods NSCs were cultured in aseptic conditions for 14-16 days embryos in vitro. Nestin immunofluorescence was used to detect NSCs. Two weeks after the induction of GDNF + IL-1α, the expression of β-tubulinⅢ and TH immunofluorescent staining was used to calculate the positive rate of each group. Fluorescent quantitative PCR was used to detect the expression of Mash1 and Hes1 genes at 1 week and 2 weeks after P4 generation of NSCs were differentiated. Results The results of immunofluorescence showed that the percentage of Nestin positive cells in NSCs was (93.04 ± 3.55)%, and the positive rate of β-tubulin Ⅲ in experimental group was (12.65 ± 1.58)% and (33.95 ± 2.97) )%, The difference between the two groups was statistically significant (P <0.05). The positive rate of TH in experimental group and control group were (1.46 ± 0.41)% and (15.51 ± 1.94)%, respectively, with statistical significance (P <0.05). The result of real-time PCR showed that the expression of Mash1 gene in NSCs was higher than that in undifferentiated state, and there was significant difference among the groups (P <0.05) (P <0.01). The expression of Hes1 gene decreased after differentiation. There was no significant difference between the control group and the experimental group and between the one week and two weeks of differentiation (P> 0.05). Conclusion The combined effect of GDNF and IL-1α can induce the differentiation of NSCs into dopaminergic neurons. Mash1 and Hes1 genes may be involved in the differentiation of NSCs into dopaminergic neurons.