两种体系下诱导多潜能干细胞定向分化为运动神经元前体细胞的差异

doi:10.3969/j.issn.1006-9771.2018.03.004

《中国康复理论与实践》 ›› 2018, Vol. 24 ›› Issue (3): 269-276.doi: 10.3969/j.issn.1006-9771.2018.03.004

两种体系下诱导多潜能干细胞定向分化为运动神经元前体细胞的差异

李哲¹, 方明珠¹, 陈红², 郭钢花¹, 范家宏¹, 毛志娟²

1.郑州大学第五附属医院,河南郑州市 450052;
2.华中科技大学同济医院,湖北武汉市430030

收稿日期:2017-11-13 修回日期:2018-01-11 出版日期:2018-03-25 发布日期:2018-03-27
通讯作者: 李哲。E-mail: lizhe.1974@163.com
作者简介:李哲(1974-),男,汉族,河南邓州市人,博士,主任医师,硕士研究生导师,主要研究方向：神经系统疾病的康复基础与临床研究。
基金资助:
1.国家自然科学基金面上项目(No. 81471302); 2.河南省科技厅基础研究计划项目(No. 142300410035)

Comparison of Inducing Pluripotent Stem Cells to Differentiate into Motor Neuron Precursor in Two Kinds of System

LI Zhe¹, FANG Ming-zhu¹, CHEN Hong², GUO Gang-hua¹, FAN Jia-hong¹, MAO Zhi-juan²

1. Department of Rehabilitation Medicine, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, China;
2. Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China

Received:2017-11-13 Revised:2018-01-11 Published:2018-03-25 Online:2018-03-27
Contact: LI Zhe. E-mail: lizhe.1974@163.com
Supported by:
Supported by National Natural Science Foundation of China (General) (No. 81471302) and Henan Science and Technology Department Basic Research Plan (No. 142300410035)

摘要/Abstract

摘要： 目的将人诱导多潜能干细胞(iPSCs)定向分化为脊髓运动神经元前体细胞(MNP),并比较在有无饲养层两种体系下的分化效率。方法分别在鼠胚胎成纤维细胞饲养层和无饲养层体系中培养人iPSCs。诱导6 d获得神经上皮前体细胞(NEP),诱导12 d获得MNP细胞。倒置显微镜下观察细胞形态变化,免疫荧光染色鉴定iPSCs、NEP、MNP标记物,实时定量聚合酶链反应检测NEP相关基因SOX1、HOXA3,MNP相关基因OLIG2、PAX6,及多能性基因SOX2、OCT4的转录水平。结果两种体系中iPSCs均表达多能性标记物,NEP及MNP均高表达神经相关标记物,低表达多能性标记物,有饲养层体系中NEP细胞SOX1、HOXA3,MNP细胞OLIG2、OCT4基因表达明显高于无饲养层,PAX6和SOX2表达无显著性差异。结论 iPSCs在两种培养体系均可有效分化为MNP细胞,在有饲养层体系中分化效率较高。

关键词: 人诱导多潜能干细胞, 运动神经元, 神经分化

Abstract: Objective To induce human-induced pluripotent stem cells (iPSCs) to differentiate into spinal motor neuron precursor (MNP) and compare the induction efficiency in systems of feeder and feeder-free. Methods iPSCs cultured on mouse feeder cells or in feeder-free condition were induced into neuroepithelial progenitors (NEP) on the sixth day and MNP on the twelveth day. Their morphology was observed under inverted microscope, and the markers of iPSCs, NEP, MNP were detected with immunofluorescence. NEP-related genes SOX1 and HOXA3, MNP-related genes OLIG2 and PAX6, and pluripotency genes SOX2 and OCT4 were detected with real-time quantitative polymerase chain reaction. Results iPSCs expressed pluripotency markers, while NEP and MNP expressed high levels of neural related markers and low levels of pluripotency markers in two systems. The expression of the genes SOX1, HOXA3, OLIG2 and OCT4 was higher in the feeder system, and there was no significant difference in the expression of genes SOX2 and PAX6.Conclusion iPSCs can differentiate into MNP in culture systems of feeder and feeder-free, and the induction efficiency is higher in the feeder system.

Key words: human-induced pluripotent stem cells, motor neurons, neural differentiation

中图分类号:

R741.05

李哲, 方明珠, 陈红, 郭钢花, 范家宏, 毛志娟. 两种体系下诱导多潜能干细胞定向分化为运动神经元前体细胞的差异[J]. 《中国康复理论与实践》, 2018, 24(3): 269-276.

LI Zhe, FANG Ming-zhu, CHEN Hong, GUO Gang-hua, FAN Jia-hong, MAO Zhi-juan. Comparison of Inducing Pluripotent Stem Cells to Differentiate into Motor Neuron Precursor in Two Kinds of System[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2018, 24(3): 269-276.

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两种体系下诱导多潜能干细胞定向分化为运动神经元前体细胞的差异

Comparison of Inducing Pluripotent Stem Cells to Differentiate into Motor Neuron Precursor in Two Kinds of System

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