中国组织工程研究 ›› 2013, Vol. 17 ›› Issue (14): 2592-2596.doi: 10.3969/j.issn.2095-4344.2013.14.018

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

无血清神经球培养下神经干细胞的生长与分化

赵际童,沈  强,黄  斐   

  1. 上海交通大学附属上海市第一人民医院骨科,上海市  200080
  • 收稿日期:2012-04-25 修回日期:2012-06-14 出版日期:2013-04-02 发布日期:2013-04-02
  • 通讯作者: 沈强,博士,主任医师,教授,硕士生导师,上海交通大学附属上海市第一人民医院骨科,上海市 200080 shmail1231@yahoo.com
  • 作者简介:赵际童★,男,1985年生,江苏省太仓市人,上海交通大学医学院在读硕士,主要从事神经干细胞与脊髓损伤治疗的研究。 zhaoli383@ sohu.com

Growth and differentiation of neural stem cells from neurospheres after culture in serum-free medium

Zhao Ji-tong, Shen Qiang, Huang Fei   

  1. Department of Orthopedics, First People’s Hospital of Shanghai, Shanghai Jiao Tong University, Shanghai  200080, China
  • Received:2012-04-25 Revised:2012-06-14 Online:2013-04-02 Published:2013-04-02
  • Contact: Shen Qiang, M.D., Chief physician, Professor, Master’s supervisor, Department of Orthopedics, First People’s Hospital of Shanghai, Shanghai Jiao Tong University, Shanghai 200080, China shmail1231@ yahoo.com
  • About author:Zhao Ji-tong★, Studying for master’s degree, Department of Orthopedics, First People’s Hospital of Shanghai, Shanghai Jiao Tong University, Shanghai 200080, China zhaoli383@ sohu.com

摘要:

背景:体外分离培养纯度高、活力强、生物特性均一的神经干细胞并掌握其生物学特性是神经干细胞移植研究的前提。
目的:建立大鼠神经干细胞的分离、培养、纯化方法,进行细胞形态学观察、表面标志物鉴定及多向分化能力检测。
方法:利用无血清悬浮神经球方法分离培养胚胎来源的神经干细胞,进行形态学观察,用免疫细胞化学方法检测神经干细胞及其分化细胞的标志物,流式细胞仪检测细胞标志物及其比例。
结果与结论:无血清悬浮神经球培养下大鼠神经干细胞生长稳定,操作简单,可大量分离、纯化、扩增神经干细胞,所获细胞具有神经干细胞的一般生物学特性,流式细胞仪检测第3代神经干细胞巢蛋白达91.5%,并具有多向分化潜能,免疫化学染色及流式细胞仪示神经干细胞可分化形成神经元,神经胶质细胞和少突胶质细胞。

关键词: 干细胞, 干细胞培养与分化, 神经干细胞, 神经球, 细胞培养, 无血清, 悬浮, 流式细胞技术, 干细胞图片文章

Abstract:

BACKGROUND: In vitro isolation and culture of neural stem cells with high purity, strong viability and homogeneous biological characteristics are the basis of neural stem cell transplantation.
OBJECTIVE: To establish a method of isolation, cultivation and purification of rat neural stem cells in vitro, to observe cell morphology, and to assess surface markers and multi-directional differentiation capacity.
METHODS: Neural stem cells were isolated from rat embryonic cerebral cortex and cultured in serum-free medium. Immunocytochemical staining was used to examine the markers of neural stem cells and their differentiated cells. Cell surface markers were assessed by flow cytometry.
RESULTS AND CONCLUSION: Neural stem cells grew as free-floating neurospheres in the serum-free conditions.This method is simple and can isolate, purify and amplify neural stem cells in vitro. The obtained cells have the general biological characteristics of neural stem cells, accounting for 91.5% by flow cytometry at passage 3, and also have multi-directional differentiation potential. Immunocytochemical staining and flow cytometry demonstrated that neural stem cells can differentiate into neurons, astrocytes and oligodendrocytes.

Key words: stem cells, stem cell culture and differentiation, neural stem cells, neurospheres, cell culture, serum-free, suspension, flow cytometry, stem cell photographs-containing paper

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