中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (36): 6707-6710.doi: 10.3969/j.issn.1673-8225.2010.36.013

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

胚胎大鼠神经干细胞双向凝胶电泳方法的建立

刘 欢1，黄国伟1，何 冰2，杨 阳1，赵 琳1   

  1. 1天津医科大学公共卫生学院，天津市 300070；2武警医学院天津市职业与环境危害生物标志物重点实验室，天津市 300162
  • 出版日期:2010-09-03 发布日期:2010-09-03
  • 通讯作者: 黄国伟，博士，教授，博士生导师，天津医科大学公共卫生学院，天津市 300070 guoweihuang@yahoo.com.cn
  • 作者简介:刘欢★，女，1980年生，天津市人，汉族，2003年天津医科大学毕业，硕士，讲师，主要从事营养与慢性病的研究。liuhuan@tijmu.edu.cn
  • 基金资助:

    国家自然科学基金资助项目(30771797)，项目名称：叶酸对神经干细胞增殖分化作用的蛋白质组学的研究；天津医科大学科学基金(2008Ky12)，项目名称：同型半胱氨酸对大鼠学习记忆及ERK信号通路的影响。

Establishment of two-dimensional gel electrophoresis technique for embryonic rat neural stem cells

Liu Huan1, Huang Guo-wei1, He Bing2, Yang Yang1, Zhao Lin1   

  1. 1 School of Public Health, Tianjin Medical University, Tianjin  300070, China; 2 Tianjin Key Laboratory for Biomarkers of Occupational and Environmental Hazard, Medical College of Chinese People’s Armed Police Forces, Tianjin  300162, China
  • Online:2010-09-03 Published:2010-09-03
  • Contact: Huang Guo-wei, Doctor, Professor, Doctoral supervisor, School of Public Health, Tianjin Medical University, Tianjin 300070, China guoweihuang@yahoo.com.cn
  • About author:Liu Huan★, Master, Lecturer, School of Public Health, Tianjin Medical University, Tianjin 300070, China liuhuan@tijmu.edu.cn
  • Supported by:

    the National Natural Science Foundation of China, No. 30771797*; the Science Program of Tianjin Medical University, No. 2008Ky12*

PDF

345

被引次数

9

摘要：

背景：神经干细胞是当今研究热点，双向电泳是蛋白质组学研究的技术基础，有必要建立稳定的神经干细胞蛋白质组双向凝胶电泳技术体系。
目的：通过对神经干细胞双向凝胶电泳几个关键步骤的分析，建立稳定、重复性好的神经干细胞双向凝胶电泳的分离方法。
方法：采用无血清体外细胞培养方法，培养胚胎大鼠神经干细胞并进行鉴定。提取样品蛋白并裂解，被动水化，采用不同IEF参数，使用PDQuest8.0软件对图谱进行分析和比较。观察不同等点聚焦参数下双向电泳图谱质量，蛋白质点的数目及重复性。
结果与结论：不同IEF参数条件下的双向电泳图谱的质量有所不同，增加低电压的除盐步骤有助于去除电泳胶图上的横纹。在80 000 Vh聚焦总能量下，获得较理想的电泳图。通过选用适当的IEF参数，建立了适当的神经干细胞双向凝胶电泳方法。

关键词: 胚胎大鼠, 神经干细胞, 蛋白质组学, 双向凝胶电泳, 神经退行性疾病

Abstract:

BACKGROUND: Neural stem cells are the current hot topic, and two-dimensional electrophoresis is the basic technique for proteome, it is necessary to establish a stable two-dimensional gel electrophoresis technology system for neural stem cells.
OBJECTIVE: To establish a stable, reproducible two-dimensional gel electrophoresis separation method for neural stem cells through analysis of several key steps of two-dimensional electrophoresis.
METHODS: Embryonic rat neural stem cells were cultured in serum-free cell culture. Proteins were extracted from samples and lysed. After passive rehydration, samples were isolated by different isoelectric focusing (IEF) parameters. Protein maps were analyzed by PDQuest8.0. Quality of two-dimensional gel electrophoresis maps under different IEF parameters, number and reproducibility of protein spots were detected.
RESULTS AND CONCLUSION: Quality of two-dimensional gel electrophoresis diagrams differs with IEF parameters. Low-voltage step could remove the cross striations. Ideal electrophoresis could be obtained under 80 000 Vh. Proper two-dimensional gel electrophoresis methods can be constructed by optimizing IEF parameters.

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