Effect of purmorphamine on neural stem cell differentiation and its mechanism

doi:10.3969/j.issn.2095-4344.0626

Abstract

Abstract:

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 (1 μmol/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.

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, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Huang Zhi, Zhang Liang-ming, Chen Rui-qiang, Yang Yang, Yang Bu, Liu Chang, Chen Zhen-xiang, Liu Can,Rong Li-min, Liu Bin. Effect of purmorphamine on neural stem cell differentiation and its mechanism[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(29): 4675-4680.

Figures/Tables 1

2.1 神经干细胞的培养、分化及鉴定结果原代神经干细胞增殖迅速，聚集呈球形或不规则形状。传代后，杂质细胞减少，神经球呈规则圆形，折光性强，内部结构清晰。用第3代神经干细胞标志物巢蛋白Nestin进行染色，结果显示神经球内大多数细胞呈Nestin染色阳性(图1)。使用去除表皮生长因子、碱性成纤维细胞生长因子的Neural basal培养基，将生长良好的神经球贴壁培养，第2天便能看见神经球边缘有细胞密集爬出(图2A，D)，实验组神经球周围爬出的细胞数量更多更密集；第7天镜下可见神经球周围长出密密麻麻的神经细胞，并可见明显的胞体及突起(图2B，E)，实验组神经细胞生长更加良好，数量更多；第14天神经细胞数量增多，突起交织呈网络状(图2C，F)，实验组中神经细胞数量更多，交联更加紧密。 2.2 免疫荧光检测神经细胞标志物的表达对照组及实验组分别用未成熟神经元细胞标志物TUJ1、星形胶质细胞标志物GFAP、成熟神经元标志物MAP2、少突胶质细胞标志物MBP进行染色，均呈阳性，证明了神经干细胞向星形胶质细胞、少突胶质细胞及神经元分化(图3，4)。从显微镜下的荧光强度来看，第7天时实验组的TUJ1、GFAP、MAP2、MBP阳性细胞数量及神经突起均比对照组多，说明了添加purmorphamine分化培养7 d后，分化细胞中的TUJ1、GFAP、MAP2、MBP表达量较对照组明显提高；第14天时实验组的GFAP、MAP2、MBP阳性细胞数量及神经突起均比对照组多，说明了添加purmorphamine分化培养14 d后，分化细胞中的GFAP、MAP2、MBP表达量较对照组明显提高。因为TUJ1为幼稚神经元标记物，在第14天时表达量显著降低，故两组没有明显差异。通过人工结合软件计数，可知第7天对照组中TUJ1、GFAP、MAP2、MBP阳性细胞率分别为27.59%，19.00%，12.23%，5.78%；实验组的阳性细胞率分别为41.36%，23.41%，17.65%，8.82%。第14天对照组中TUJ1、GFAP、MAP2、MBP阳性细胞率分别为11.52%，18.73%，7.35%，6.52%；实验组的阳性细胞率分别为10.37%，25.17%，35.53%，10.46%。 2.3 RT-qPCR检测Shh信号通路及分化标志物表达无论是第7天还是第14天，实验组的SMO、PTCH1、GLI1表达量均比对照组显著增高(P < 0.05)，表明purmorphamine有效激活了Shh信号通路。在神经细胞分化标志物表达上，第7天时实验组的TUJ1、GFAP、MAP2、MBP表达量均比对照组显著增高(P < 0.05)，证明purmorphmine能有效促进神经干细胞向各类神经细胞分化。第14天时实验组的GFAP、MAP2、MBP表达量也显著增高；TUJ1表达量差异无显著性意义，因为TUJ1是神经元早期表达的标记物，在第14天时表达量显著减少。以上数据表明了purmorphmine能有效上调Shh信号通路表达及促进神经干细胞的分化(图5)。"

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