上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (10): 1500-.doi: 10.3969/j.issn.1674-8115.2011.10.032

• 技术与方法 • 上一篇    下一篇

少突胶质前体细胞分离培养方法的改进及其谱系限定细胞体外分化模型的建立

李 莹, 富赛里, 马政文   

  1. 上海交通大学基础医学院神经生物学实验室, 上海 200025
  • 出版日期:2011-10-28 发布日期:2011-10-27
  • 通讯作者: 马政文, 电子信箱: mazhengwen@hotmail.com。
  • 作者简介:李 莹（1980—）, 女, 技士, 本科；电子信箱: cher125ly@hotmail.com。
  • 基金资助:

    上海市教委基金（06BZ006）；上海交通大学基础医学院青年教师科研能力提升计划

Improved isolation and culture method of oligodendrocyte precursor cells and in vitro model establishment of lineage restricted oligodendrocyte differentiation

LI Ying, FU Sai-li, MA Zheng-wen   

  1. Laboratory of Neurobiology, Basic Medical College, Shanghai Jiaotong University, Shanghai 200025, China
  • Online:2011-10-28 Published:2011-10-27
  • Supported by:

    Shanghai Education Committee Foundation, 06BZ006；Foundation of Basic Medical College, Shanghai Jiaotong University

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摘要：

目的 对少突胶质前体细胞（OPCs）的分离、培养及传代方法进行改良，并建立少突胶质谱系限制细胞的体外分化模型，以模拟体内OPCs的发育过程。方法 通过非特异性差异黏附法与A2B5-IgM抗体免疫筛选法结合，从胚胎大鼠脊髓来源的细胞悬液中分离、纯化OPCs，用含成纤维细胞生长因子-2和血小板源性生长因子的培养液作扩增培养；再以三碘甲状腺氨酸和神经营养因子-3诱导OPCs分化，最终分化为成熟的少突胶质细胞（OLs）。OPCs及其分化细胞的特性通过形态学观察和免疫化学染色法鉴定。结果 95%以上的细胞具有双极或三极突起的典型OPCs形态，并表达A2B5；在一定的培养条件下，OPCs可持续扩增和反复传代，且扩增的OPCs仍能维持其特有的形态和自我增殖的特性；进入分化进程的细胞将依次表达O4、O1、受体反应蛋白及髓鞘碱性蛋白等特异性分化抗原。结论 分离的OPCs在形态、增殖以及分化格局等方面均与存在于胚胎脑区的O2A前体细胞（体内OPCs）相类似。

关键词: 少突胶质前体细胞, 杂交瘤细胞株, 成纤维细胞生长因子-2, 血小板源性生长因子, 细胞培养

Abstract:

Objective To obtain healthier and purer oligodendrocyte precursor cells (OPCs) via modification of the methods in OPCs isolation, culture and generation passage, and to mimic the development of OPCs in vivo via setup of an in vitro model of lineage restricted oligodendrocyte differentiation. Methods Using nonspecific differentiated pre-attachment and A2B5IgM immuno-panning, OPCs isolated from embryonic rat spinal cords were highly purified. Their proliferations were induced by enrichment of the culture solution with fibroblast growth factor-2 and platelet-derived growth factor, and their differentiations to mature oligodendrocytes were induced with tri-iodothyronine and neurotrophin-3. The morphology and characteristics of OPCs and cells differentiated from them were observed using microscope and immunostaining. Results More than 95% of the cells exhibited typical OPCs morphology of bipolar or tripolar processes, and expressed A2B5. These cells could proliferate steadily and execute generation passage repeatedly. After inducement into differentiation process, cells expressed specific antigens, such as O4, O1, receptor-interacting protein and myelin basic protein, sequentially. Conclusion The morphology, proliferation and differentiation of the isolated OPCs are similar with the O2A precursor cells in embryonic brain (natural OPCs).

Key words: oligodendrocyte precursor cell, hybridoma cell line, fibroblast growth factor-2, platelet-derived growth factor, cell culture