中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (9): 1330-1335.doi: 10.3969/j.issn.2095-4344.1592

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

贴壁培养法鉴定海马神经干/祖细胞的增殖与分化能力

胡玉婷，刘 菲，刘 静，陶欣荣   

  1. 安徽理工大学医学院，安徽省淮南市 232001
  • 修回日期:2018-11-15 出版日期:2019-03-28 发布日期:2019-03-28
  • 通讯作者: 陶欣荣，博士，教授，安徽理工大学医学院，安徽省淮南市 232001
  • 作者简介:胡玉婷，女，1994年生，安徽省六安市人，汉族，安徽理工大学在读硕士，主要从事尼古丁发育毒性研究。
  • 基金资助:

    国家自然科学基金面上项目(81471161)，项目负责人：陶欣荣；安徽省RC18-学科(专业)拔尖人才(gxbjZD16)，项目负责人：陶欣荣

Identification of proliferation and differentiation capability of hippocampal neural stem/progenitor cells by adherent culture

Hu Yuting, Liu Fei, Liu Jing, Tao Xinrong   

  1. School of Medicine, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China
  • Revised:2018-11-15 Online:2019-03-28 Published:2019-03-28
  • Contact: Tao Xinrong, MD, Professor, School of Medicine, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China
  • About author:Hu Yuting, Master candidate, School of Medicine, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81471161 (to TXR); Anhui Province RC18-Discipline (Professional) Top Talents, No. gxbjZD16 (to TXR)

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摘要：

文章快速阅读：

文题释义：
神经干/祖细胞：指可保持自我更新、分化为不同类型神经细胞的前体细胞，可划分为几种类型，1型代表位于成体脑齿状回的放射状静止干细胞，GFAP和Nestin表达均阳性，并存在星形胶质细胞的表征；2型即GFAP阴性和Nestin阳性具有高度增殖特征的细胞；3型指神经母细胞，DCX阳性和Nestin阴性；其中1型细胞被定义为神经干细胞，而2型和3型被定义为瞬时放大神经祖细胞。
细胞贴壁培养：指的是细胞黏附在某一固体表面展开的培养，贴壁扩散形成单层。悬浮细胞若贴壁培养需要进行处理，例如提前对培养皿进行多聚赖氨酸包被，培养皿表面带有多聚阳离子分子，从而促进细胞黏附。

 

摘要
背景：神经干/祖细胞广泛应用于神经发育、神经相关性疾病和细胞移植等研究，体外分离培养的神经干/祖细胞能够为相关研究提供细胞模型。
目的：探讨分离新生小鼠海马组织并高效培养出神经干/祖细胞的方法，并使用贴壁培养法对其增殖以及分化能力进行鉴定。
方法：分离新生C57BL/6小鼠海马组织，培养原代神经干/祖细胞并扩增，进行形态学观察；联合使用多聚赖氨酸和层粘连蛋白促进细胞贴附，进行贴壁培养；免疫荧光法检测特异性标志蛋白Nestin表达和增殖指标Ki-67的表达；使用神经干/祖细胞分化培养基诱导分化培养7 d后，免疫荧光法检测GFAP、DCX、Tuj1和S100β的表达。
结果与结论：①培养的神经干/祖细胞约82%表达Nestin，约49%表达Ki-67；②诱导分化后，少数细胞为DCX阳性，大多数细胞为GFAP阳性。荧光双色染色后显示Tuj1和S100β表达的神经元和星形胶质细胞比例为1∶1.7；③结果显示，从新生C57BL/6小鼠海马组织中成功分离并高效培养出神经干/祖细胞，贴壁培养后有效地对其增殖和分化能力进行了鉴定，能够为进一步实验研究提供足够的细胞来源。


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
ORCID: 0000-0002-2106-6039(陶欣荣)

关键词: 神经干/祖细胞, 海马, 悬浮培养法, 贴壁培养法, 干细胞, 国家自然科学基金

Abstract:

BACKGROUND: In vitro isolation and culture of neural stem/progenitor cells will provide a good cell model for the study of neurodevelopment, neurological diseases, and neural transplantation. 
OBJECTIVE: To study the highly effective method for isolation and expansion of hippocampal neural stem/progenitor cells from newborn mice, and to identify the proliferation and differentiation of hippocampal neural stem/progenitor cells using improved adherent culture method.
METHODS: Neural stem/progenitor cells were isolated from the hippocampus of newborn C57BL/6 mice and were expanded for several passages. Combination of polylysine and laminin were used for adherent culture to promote cell attachment. Morphological observation and immunofluorescence cytochemical staining were conducted to detect the expression of neural progenitor-specific marker protein Nestin and proliferation index Ki-67. After 7 days of induction and differentiation, the expression of GFAP, DCX, Tuj1 and S100β was detected by immunofluorescence.
RESULTS AND CONCLUSION: About 82% of the cultured neural stem/progenitor cells expressed Nestin, and about 49% expressed Ki-67. A small number of cells were DCX-positive neurons after induction and differentiation, while most of the cells were positive for GFAP. The ratio of neurons to astrocytes was 1:1.7 identified by Tuj1 and S100β double staining. The neural stem/progenitor cells derived from the hippocampus were efficiently isolated and cultured. The cell proliferation and differentiation abilities were effectively identified after adherent culture, which can provide sufficient cell sources for further experimental research.


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, Hippocampus, Tissue Engineering

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