Growth and differentiation of neural stem cells on a keratin fiber scaffold carrying nerve growth factor

doi:10.3969/j.issn.2095-4344.0533

Abstract

Abstract:

BACKGROUND: It is essential for nerve tissue engineering is to construct a three-dimensional biomaterial scaffold carrying nerve cells, which has huge potential for repair of injured nervous system.
OBJECTIVE: To seek the optimal concentration of hydrochloric acid used to treat keratin fibers based on the physical and chemical properties of keratin fibers, and to explore the effect of keratin fibers carrying nerve growth factor (NGF) on the differentiation of neural stem cells.
METHODS: Keratin fibers were treated by hydrochloric acid of various concentrations, and their chemical properties were anlayzed by Fourier transform infrared spectroscopy. Then, we determined the optimal concentration of hydrochloric acid to treat keratin fibers. Subsequently, NGF was loaded onto the keratin fibers as shown by the Fourier transform infrared spectroscopy. Neural stem cells were co-cultured with keratin fibers with or without NGF for 10 days, and the expression of MAP2, MBP, GFAP and the percentage of nerve cells were thereafter detected by immunofluorescence.
RESULTS AND CONCLUSION: The highest content of exposed carboxyl on the fiber surface appeared after treatment of keratin fibers with 8 mol/L hydrochloric acid. After 10 days of co-culture, the percentage of nerve cells was significantly higher in the keratin fiber-NGF group than the keratin fiber and control groups. Findings from the present study indicate that co-culture with keratin fibers carrying NGF can dramatically increase the percentage of nerve cells differentiating from neural stem cells.

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: Keratins, Nerve Growth Factor, Neural Stem Cells, Neurons, Tissue Engineering

CLC Number:

R394.2

Sun Quan, Li Ying, Gao Yu-dan, Hao Peng, Zhao Wen, Duan Hong-mei, Yang Zhao-yang, Li Xiao-guang . Growth and differentiation of neural stem cells on a keratin fiber scaffold carrying nerve growth factor[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3387-3392.

Figures/Tables 2

2.1 红外光谱不同浓度盐酸处理后的角蛋白纤维均在红外光谱谱图中显现出了羧基的特征峰。在3 289 cm-1处的大而宽的峰是-COOH中-OH的伸缩振动峰。随着盐酸浓度的增加，-OH的峰值和峰面积都出现增加(图1A)。8 mol/L盐酸处理后的角蛋白纤维(K8)表现出最高的羧基含量，所以之后的实验选择K8进行。NGF的红外光谱谱图中表现出了氨基的特征峰，其中3 409 cm-1是游离的-NH的伸缩振动峰，1 464 cm-1是N-H的弯曲振动峰。K8的红外光谱谱图中表现出了羧基的特征峰。在K8-NGF的红外光谱谱图中，3 317 cm-1是酰胺键中-NH的伸缩振动峰，这代表成功进行了酰胺反应，NGF加载到了纤维上(图1B)。 2.2 神经球与材料共培养后的细胞形态 K8组和对照组在共培养后1 d，仅有少量的神经球黏附到培养皿底部，大部分神经球没有突起伸展出来。随着培养时间的延长，到第7天，少量的细胞从神经球中迁移出来，迁移出的细胞胞体扁平，突起较短。光镜下，K8组和对照组相比细胞形态无明显差异。K8-NGF组在共培养第1天，可以观察到大量的神经球贴壁，黏附在培养皿底部，同时有少量短的突起从贴壁神经球中伸展出来。共培养第7天，可以观察到大量的细胞从神经球中迁移分化出来，与共培养第1天相比，突起的长度明显增长，细胞之间形成复杂的细胞网络，见图2。 2.3 神经突起的定量分析结果共培养第1天，对照组和K8组只有很短的突起伸出，分别为(26.96±11.31) μm和(22.31±8.39) μm。第5天，对照组和K8组突起变长，且两组没有显著性差异，分别为(74.46±18.84) μm和(72.49± 16.87) μm。K8-NGF组在第1天共培养后就有大量短的突起从神经球中伸展出来，达(106.60±18.25) μm，且随着培养时间的延长突起的长度继续增加。在第7天，K8-NGF组中从神经球伸展出的突起长度为(279.09±36.45) μm，明显长于其他两组，见图3。 2.4 细胞活性共培养第1，3，7天，K8组和对照组的吸光度值在各时间点均无显著性差异，K8-NGF组吸光度值明显高于其他两组。共培养第1天到第3天，对照组和K8组的吸光度值都有所增加，但是从第3天到第7天对照组和K8组的吸光度值都基本没有变化。K8-NGF组一直保持很稳定的高细胞活性状态，见图4。 2.5 角蛋白纤维和角蛋白纤维-NGF对NSCs分化的影响分别用神经元标记物MAP-2，星形胶质细胞标记物GFAP，少突胶质细胞标记物MBP进行免疫荧光染色，以观察共培养后NSCs的分化及比例。使用ImageJ进行细胞计数。结果显示，共培养10 d后，K8-NGF组的大部分NSCs分化成了神经元(80.78±1.95)%，少部分分化成了星形胶质细胞和少突胶质细胞，分化的细胞表型比例为神经元>星形胶质细胞>少突胶质细胞，其中分化为神经元的比例显著高于对照组和K8组。对照组和K8组中的NSCs也分化成了神经元、星形胶质细胞和少突胶质细胞，但是细胞表型的比例为星形胶质细胞>神经元>少突胶质细胞，仅有少量分化为了神经元[(31.65±2.18)%和(33.93±2.92)%]，K8组和对照组分化出的各种细胞表型的比例均无显著性差异，见图5和图6。"

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