基础医学与临床 ›› 2012, Vol. 32 ›› Issue (1): 83-86.

• 技术与方法 • 上一篇    下一篇

新生大鼠海马神经元原代无血清培养与鉴定

黄立宁1,韩建民1,刘雅2,刘悦1,曹翠丽3   

  1. 1. 河北医科大学第二医院麻醉科
    2. 河北医科大学第二医院
    3. 河北医科大学基础所细胞生物室
  • 收稿日期:2011-05-20 修回日期:2011-08-22 出版日期:2012-01-05 发布日期:2011-12-28
  • 通讯作者: 曹翠丽 E-mail:caocuili615@163.com

Serum-free Primary Culture and Identification of Hippocampal Neurons from Newborn Rats

  • Received:2011-05-20 Revised:2011-08-22 Online:2012-01-05 Published:2011-12-28

摘要: 摘要:目的 建立一种简单、易行的海马神经元无血清体外培养方法以获得高纯度、高活力的海马神经元。方法 取新生24h内SD大鼠,分离海马,经消化后种植于包被有Matrigel基膜的玻片上,24h后换含有N2、B27的neurobasal无血清培养基,于不同时间在倒置相差显微镜下观察细胞的生长状态;采用β-tublinⅢ免疫荧光细胞化学技术鉴定海马神经元纯度。结果 大部分海马神经元于3-24h贴壁且可长出细长突起,3d细胞具有典型神经元的形态特征, 5d神经突起进一步增多并形成稠密的网络连接,7d神经元胞体丰满,趋于成熟。经β-tublinШ免疫荧光技术鉴定纯度为(94.2±3.6)%。结论 此方法简单有效,可获得高纯度的海马神经元。

关键词: 关键词: 海马, 神经元, 原代培养, 新生大鼠

Abstract: Abstract: Objective To establish a simple and practical method of the serum-free primary culture of hippocampal neurons in vitro to obtain highly purified and energetic neurons. Methods Hippocampus of newborn rats(24 hours) were taken out and digested. Hippocampal neurons were planted in the glass slides covered with Matrigel basement membrane. Twenty-four hours after the cell being plated, the culture medium was removed and replaced by serum-free neurobasal one with N2 and B27 supplementations. The morphological changes of the neurons were observed under inverted phase-contrast microscope at different time. Immunofluorescence staining for β-tublinⅢ was performed to identify the purity of neurons. Results A large number of hippocampal neurons began to adhere to the glass slides and develop small neurites in 3-24 hours. Then, a typical neuronalmorphology appeared on the third day. Up to the 5th day, many neurites extended to form dense network. Soma of neurons became were well developed on the 7th day. Fluorescence staining with β-tublinⅢ showed that the purity of neurons was 94.2±3.6%. Conclusion The hippocampal neurons with high purity can be obtained by the simple and efficient method.

Key words: Key words: hippocampus, neurons, primary culture, newborn rats

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