Purmorphamine对神经干细胞分化的影响及其机制

Effect of purmorphamine on neural stem cell differentiation and its mechanism

背景:神经干细胞的增殖及分化一直是研究的难点和热点,有效提高神经干细胞向神经细胞的分化效率具有重要的意义。据最近文献报道,小分子化合物purmorphamine在各类细胞增殖及分化实验中的应用越来越广泛。目的:探讨小分子化合物purmorphamine通过上调Shh信号通路对神经干细胞分化为各类神经细胞的促进作用。方法:从小鼠胚胎海马区分离培养得到原代神经干细胞,取第2-4代生长良好的细胞分为对照组及实验组。对照组用不添加purmorphamine的分化培养基培养,实验组用添加1μmol/L purmorphamine的分化培养基培养。培养7,14 d后,通过免疫细胞化学、RT-qPCR方法检测神经细胞标志物的表达量。结果与结论:(1)体外培养的神经干细胞生长良好,神经球中的大部分细胞表达特异性标志物nestin;(2)对照组及实验组均可见到TUJ1、GFAP、MAP2、MBP染色阳性;(3)RT-qPCR显示第7天实验组的TUJ1、GFAP、MAP2、MBP表达量均比对照组显著增高(P<0.05);第14天实验组的Smo、Ptc1、Gli-1表达量比对照组显著增高(P<0.05);GFAP、MAP2、MBP的表达量也比对照组显著增高(P<0.05);(4)以上数据表明purmorphmine能通过上调Shh信号通路来促进神经干细胞的分化。

BACKGROUND:The proliferation and differentiation of neural stem cells have always been a difficulty and a hot topic,which is of great significance to effectively increase the differentiation efficiency of neural stem cells into nerve cells.Recent reports have shown that purmorphamine,a small molecule compound,is widely used in various cell proliferation and differentiation experiments.OBJECTIVE:To investigate the role of purmorphamine in promoting the differentiation of neural stem cells into various types of nerve cells by up-regulating the Sonic Hedgehog(Shh)signaling pathway.METHODS:Primary neural stem cells were isolated from the hippocampus of mouse embryonic hippocampus.Cells at passages 2-4 were cultured in differentiation medium with purmorphamine(1μmol/L;experimental group)or without purmorphamine(control group)for 7 and 14 days.The differentiation of neural stem cells was detected by immunocytochemistry and RT-qPCR.RESULTS AND CONCLUSION:Neural stem cells grew well in vitro,and most of the cells in the neurospheres expressed a specific marker of nestin.The cells cultured in the two groups were positive for TUJ1,GFAP,MAP2 and MBP.RT-qPCR results showed that the expression of TUJ1,GFAP,MAP2 and MBP in the experimental group was significantly higher than that in the control group at 7 days of culture(P<0.05).The expression of Smo,Ptc1 and Gli-1 in the experimental group was significantly higher than that in the control group and the expression of GFAP,MAP2 and MBP was also significantly increased at 14 days of culture(P<0.05).To conclude,purmorphmine can promote the differentiation of neural stem cells by up-regulating the Shh signaling pathway.