Armstrong R C
Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Methods. 1998 Nov;16(3):282-92. doi: 10.1006/meth.1998.0685.
Using in vitro systems, the proliferation, migration, differentiation, and survival of immature oligodendrocyte lineage cells can be examined to elucidate the cellular and molecular interactions that regulate this lineage. The ability to monitor progressive stages of differentiation within the lineage by immunophenotyping and to manipulate the cellular responses with growth factors makes these cultures advantageous as both a method for studying the cell biology of myelination and as a model system for lineage analysis in the mammalian central nervous system. In addition, cultured oligodendrocytes carry out the normal in vivo sequence of expression of a set of cell type-specific genes, some of which are extremely highly expressed, and so provide advantages for analysis of gene regulation. This paper describes commonly used methods for the preparation of mixed glial cell cultures from perinatal rodent brain. Although these cultures are most commonly derived from perinatal rat brain, a protocol for preparation from mouse brain is also provided because of the increasing number of studies that use mice to facilitate molecular biological techniques. Methods to prepare secondary cultures of different stages of oligodendrocyte lineage cells are detailed. As examples of methods to use for the characterization of these cells, immunophenotypes of each stage of the oligodendrocyte lineage are illustrated, incorporation of [3H]thymidine for analysis of cell proliferation is illustrated, and detailed methods are provided for analysis of migration in a microchemotaxis chamber.
利用体外系统，可以检测未成熟少突胶质细胞谱系细胞的增殖、迁移、分化和存活情况，以阐明调节该谱系的细胞和分子相互作用。通过免疫表型分析监测谱系内分化的进展阶段以及用生长因子操纵细胞反应的能力，使这些培养物成为研究髓鞘形成细胞生物学的方法以及哺乳动物中枢神经系统谱系分析模型系统的优势所在。此外，培养的少突胶质细胞会按照体内正常顺序表达一组细胞类型特异性基因，其中一些基因表达水平极高，因此为基因调控分析提供了优势。本文描述了从围产期啮齿动物脑制备混合胶质细胞培养物的常用方法。尽管这些培养物最常见的来源是围产期大鼠脑，但由于越来越多的研究使用小鼠来促进分子生物学技术，因此也提供了从小鼠脑制备的方案。详细介绍了制备不同阶段少突胶质细胞谱系细胞二级培养物的方法。作为用于表征这些细胞的方法示例，展示了少突胶质细胞谱系每个阶段的免疫表型，展示了用于分析细胞增殖的[3H]胸腺嘧啶核苷掺入情况，并提供了在微趋化室中分析迁移的详细方法。
