活性生物材料支架体外诱导神经干细胞向神经元高比例分化并形成神经网络

High-proportion differentiation of neural stem cells into neurons and formation of neural networks induced by active biomaterial scaffolds in vitro

背景:活性生物材料的良好组织相容性和生物活性与神经干细胞的多向分化潜能均有极大的应用前景和应用价值。目的:观察神经营养因子3-壳聚糖活性生物材料支架在体外对神经干细胞的诱导分化作用及神经干细胞神经营养因子3信号通路关键蛋白的表达。方法:提取并纯化新生24 h Wistar大鼠脊髓神经干细胞,分4组进行诱导分化:对照组(单纯培养基组)、可溶性神经营养因子3组、单纯壳聚糖组、神经营养因子3-壳聚糖组。诱导6 h,提取蛋白质行Western Blot检测神经营养因子3信号通路关键蛋白TrkC、Akt/p-Akt、Erk/p-Erk的表达;诱导7 d,行MAP2、MBP、GFAP免疫细胞化学染色观察神经元的多向分化及各类细胞的分化比例;诱导14 d,行MAP2、Synapsin-1、PSD95免疫细胞化学染色观察神经营养因子3-壳聚糖活性生物材料是否诱导神经干细胞分化形成神经网络。结果与结论:(1)神经营养因子3-壳聚糖组诱导神经干细胞高比例分化为神经元,比例为73.8%,明显高于其他3组,同时分化形成的胶质细胞低于其他3组;(2)神经营养因子3-壳聚糖组中关键蛋白TrkC、p-Akt、p-Erk的表达高于其他3组;(3)神经营养因子3-壳聚糖可以诱导神经干细胞在体外分化形成神经网络。

BACKGROUND:Either good biocompatibility and biological activity of active biological materials or the potential of multidirectional differentiation of neural stem cells has great application prospect and value.OBJECTIVE:To investigate the effect of neurotrophic factor 3-chitosan active biomaterial scaffolds on the differentiation of neural stem cells and the expression of key proteins of the neurotrophic factor 3 signal pathway in vitro.METHODS:The neural stem cells were extracted and purified,and then divided into pure culture medium group,soluble neurotrophic factor 3 group,pure chitosan group,and neurotrophic factor 3-chitosan group for differentiation induction.The expression of TrkC,Akt/p-Akt and Erk/p-Erk in the neurotrophic factor 3 signaling pathway was detected by western blot after 6 hours of induction.After 7 days of induction,differentiation of neural stem cells was observed by immunocytochemistry of MAP2,MBP,and GFAP.After 14 days of induction,formation of neural network induced by neurotrophic factor 3-chitosan active biomaterials was observed by immunocytochemistry of MAP2,Synapsin-1,and PSD95.RESULTS AND CONCLUSION:The neurotrophic factor 3-chitosan group induced a high proportion of neural stem cells differentiated into neurons,with a ratio of 73.8%,which was significantly higher than that in the other three groups.Meanwhile,the proportion of cells differentiated into glial cells waslower than that in the other three groups.The expression of key proteins TrkC,p-Akt and p-Erk in the neurotrophic factor 3-chitosan group was higher than that in the other three groups.Meanwhile,neurotrophic factor 3-chitosan could induce the in vitro differentiation of neural stem cells to form neural network