少突胶质前体细胞分离培养方法及其谱系限定细胞体外分化模型建立的改进

Improvement in isolation and culture method for oligodendrocyte precursor cells and establishment of the in vitro model for lineage restricted oligodendrocyte differentiation

目的:对少突胶质前体细胞(oligodendrocyte precursor cells,OPCs)的分离、培养及传代方法进行改良,探讨体外条件下OPCs的增殖与分化特性。方法:取新生SD大鼠脊髓来源的细胞分离、纯化OPCs行原代培养,用含碱性成纤维细胞生长因子(basic fibroblast growth factor,b FGF)和血小板源性生长因子-aa(platelet-derived growth factor-aa,PDGF-aa)的培养液作扩增培养,MTT法检测OPCs的增殖能力;三碘甲腺原氨酸和睫状神经生长因子诱导OPCs分化,最终分化为成熟的少突胶质细胞(oligodendrocytes,OLs),并对分化细胞行形态学及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;MTT法检测显示OPCs在体外保持良好的增殖能力;细胞最终诱导分化为成熟的OLs并表达髓鞘碱性蛋白(myelin basic protein,MBP)。结论:OPCs在体外条件下可继续保持良好的增殖生长及定向分化能力。

Objective： To establish the improved methods for oligodendrocyte precursor cells （OPCs） isolation, culture and subculture and to investigate the characteristics of OPCs differentiation and proliferation in vitro. Methods： OPCs isolated from neonatal SD rat spinal cords were cultured in vitro. Cell proliferation was induced by the culture solution with basic fibroblast growth factor and platelet-derived growth factor, and the proliferation of OPCs was determined by MTT assay. OPCs differentiation to mature oligodendrocytes was induced by triiodothyronine and ciliary neurotrophic factor. The morphology and characteristics of OPCs and differentiated cells were observed via microscope and immunostaining. Results： More than 95% of the cells exhibited typical OPCs morphology with bipolar or tripolar bumps and expressed A2B5, while the OPCs kept the proliferative capacity in vitro. After culture for differentiation, the OPCs progressively differentiated into mature oligodendrocytes which specifically expressed myelin basic protein. Conclusion： The OPCs retain the ability of differentiation and proliferation in vitro.