上海交通大学学报（医学版）

›› 2010, Vol. 30 ›› Issue (11): 1444-.doi: 10.3969/j.issn.1674-8115.2010.11.031

• 技术与方法 • 上一篇    下一篇

高纯度少突胶质前体细胞改良培养方法的建立

王兴启, 刘 轩, 杨丽华, 戚大石, 刘 静, 姚瑞芹   

  1. 徐州医学院 神经生物学教研室, 徐州 221002
  • 出版日期:2010-11-25 发布日期:2010-11-29
  • 通讯作者: 姚瑞芹, 电子信箱: wenxi_yao@163.com。
  • 作者简介:王兴启（1985—）, 男, 硕士生；电子信箱: wangjunming1161@yeah.net。
  • 基金资助:

    国家自然科学基金（30801526）；江苏省高校自然科学基金(09KJD310010)；徐州医学院院长专项基金（09KJZ14）

Improved culture method for oligodendrocyte precursor cells with high purity

WANG Xing-qi, LIU Xuan, YANG Li-hua, QI Da-shi, LIU Jing,YAO Rui-qin   

  1. Department of Neurobiology, Xuzhou Medical College, Xuzhou 221002, China
  • Online:2010-11-25 Published:2010-11-29
  • Supported by:

    National Natural Science Foundation of China, 30801526；Natural Science Foundation for Universities in Jiangsu Province, 09KJD310010；Foundation of Xuzhou Medical College, 09KJZ14

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摘要：

目的 对传统培养方法进行改良，以获得更高纯度的少突胶质前体细胞(OPCs)。方法 新生1~2 d Sprague-Dawley（SD）大鼠的大脑皮质混合胶质细胞原代培养8 d后，采用恒温摇床振荡分离法、差速贴壁法和胰酶消化法结合条件限定培养基纯化培养细胞，台盼蓝染色光学显微镜观察细胞形态；纯化培养2 d后进行A2B5和DAPI免疫荧光染色鉴定和细胞纯度分析。结果 光学显微镜观察发现纯化培养细胞的胞膜完整，细胞无损伤；免疫荧光染色后细胞纯度分析显示，A2B5染色阳性细胞占DAPI染核细胞的百分比为98.14%。结论 通过对传统的混合胶质细胞培养及振荡分离和纯化方法的改进，能够培养出高纯度的OPCs。

关键词: 少突胶质前体细胞, 细胞培养, 纯化, 鉴定, 大鼠

Abstract:

Objective To establish an improved culture method for obtaining oligodendrocyte precursor cells (OPCs) with high purity. Methods Mixed glial cells of cerebral cortex of newborn (1 to 2 d after born) Sprague-Dawley (SD) rats were primarily cultured for 8 d, and cells were cultured and purified by orbital shaking, differential adhesion and trypsin digestion in combination with defined culture media. Cell morphology was observed by light microscope with trypan blue staining. Two days after culture, cells were identified by immunofluorescence staining with A2B5 and DAPI, and purity analysis was conducted. Results It was observed by light microscopy that the cytomembrane of cultured cells was intact, with no cell injury. Purity analysis after immunofluorescence staining revealed that the ratio of number of A2B5-positive cells to that of DAPI-positive cells was 98.14%. Conclusion OPCs with high purity can be obtained by the improved methods of mixed glial cell culture and purification.

Key words: oligodendrocyte precursor cell, cell culture, purification, identification, rat