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

doi:10.3969/j.issn.2095-4344.0476

Abstract

Abstract:

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.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Neural Stem Cells, Neurotrophin 3, Chitosan, Neurons, Tissue Engineering

CLC Number:

R394.2

Li Ying, Zhang Ai-feng, Gao Yu-dan, Zhao Wen, Duan Hong-mei, Hao Peng, Shang Jun-kui, Yang Zhao-yang, Li Xiao-guang. High-proportion differentiation of neural stem cells into neurons and formation of neural networks induced by active biomaterial scaffolds in vitro[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1432-1437.

Figures/Tables 1

2.1 脊髓源神经干细胞的培养、纯化与鉴定结果神经细胞悬液接种约24 h后逐渐形成小神经球，可观察到较多的细胞碎片。随着细胞的增殖，神经球球体逐渐增大变圆，悬浮液中细胞碎片减少。传代至第3代时可见纯化的神经球体积较大、边界清晰、折光性强，纯化的神经球表达神经干细胞特异性标记物巢蛋白(Nestin)(图1)，证明提取并纯化的脊髓源性神经球为神经干细胞。 2.2 神经营养因子3-壳聚糖活性生物材料支架诱导神经干细胞高比例向神经元分化并抑制神经干细胞向胶质细胞分化诱导7 d后4组神经球均发生不同程度的分化，各组均可见微管相关蛋白2(MAP2)阳性神经元、胶质纤维酸性蛋白(GFAP)阳性星形胶质细胞、髓磷脂碱性蛋白(MBP)阳性少突胶质细胞，其中神经营养因子3-壳聚糖组神经元比例最高(73.8%)且明显高于其他3组，而星形胶质细胞和少突胶质细胞比例最低(图2)，证明了神经干细胞具有多向分化潜能，同时也证明了神经营养因子3-壳聚糖活性生物材料支架能够诱导神经干细胞向神经元高比例分化，并抑制胶质细胞的生成。 2.3 神经营养因子3-壳聚糖活性生物材料支架诱导TrkC信号通路关键蛋白高比例表达神经营养因子3主要通过与细胞膜表面神经营养因子3受体，即TrkC受体结合，进而激活Akt及MAPK/Erk通路以维持神经干细胞的生长和分化，并且介导突触的形成。通过检测神经营养因子3信号通路中TrkC受体、Akt/p-Akt、Erk/p-Erk不同时间点蛋白表达，发现在4组神经球诱导分化至6 h时，被检蛋白的表达含量最高，因此提取4组神经球诱导分化6 h时的蛋白进行Western Blot检测蛋白表达水平，发现神经营养因子3-壳聚糖组神经干细胞表达的关键蛋白水平高于其他3组(图3)，进一步证明神经营养因子3-壳聚糖活性生物材料支架能够促进神经元的生长和分化。 2.4 神经营养因子3-壳聚糖活性生物材料支架诱导神经干细胞形成突触与神经网络突触形成是神经元成熟的标志，也是神经网络形成的基础。由于神经营养因子3-壳聚糖活性生物材料支架能够诱导神经干细胞高比例分化形成神经元，因此进一步检测神经营养因子3-壳聚糖支架诱导分化出的神经元之间是否能形成突触结构。诱导14 d后对神经营养因子3-壳聚糖组的细胞进行免疫细胞化学染色发现，MAP2阳性神经元之间有突触囊泡蛋白(Synapsin-1)阳性的突触前成分表达，也有突触后密度蛋白(Postsynaptic Density Protein 95，PSD95)阳性的突触后成分表达，并且突触前成分和突触后成分存在共定位现象(图4)，说明神经营养因子3-壳聚糖活性生物材料支架诱导神经干细胞分化出的神经元之间形成了突触，同时分化出大量的神经元之间也通过突触的相互联系形成了神经网络。"

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