Proteomic mapping of oligodendrocyte-associated proteins

少突胶质细胞相关蛋白的蛋白质组图谱

• 批准号: 6763970
• 负责人: RAYMOND J COLELLO
• 金额: $ 19.7万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6763970
• 关键词: X ray; gene expression; genetic regulation; immunocytochemistry; in situ hybridization; laboratory rat; liquid chromatography mass spectrometry; matrix assisted laser desorption ionization; nerve /myelin protein; oligodendroglia; optic nerve; protein quantitation /detection; proteomics; two dimensional gel electrophoresis; western blottings

DESCRIPTION (provided by applicant): Recent studies examining the role of oligodendrocytes in development, and following disease or trauma, have exposed our limited understanding of oligodendrocyte function and underscore the need to establish new tools which will allow us to uncover the full repertoire of proteins regulated by oligodendrocytes and ascertain their function. Unfortunately, the lack of cDNA libraries derived from myelinating oligodendrocytes has hampered attempts to identify proteins derived from or regulated by these cells. We therefore established a new method to elucidate such proteins by coupling the techniques of X-irradiation, 2-D gel electrophoresis, and mass spectrometry. Specifically, in previous studies, we had demonstrated that oligodendrocytes could be selectively eliminated from one optic nerve of a rat by treating the animal to a unilateral exposure of X-irradiation at the time of birth. We further showed that, with time, the X-irradiated optic nerve became repopulated with a second cohort of oligodendrocytes from postnatal day (P) 14 on. This repopulation of cells into the X-irradiated nerve nearly restores the full complement of oligodendrocytes normally found in the optic nerve by P28. Therefore, utilizing this approach, we experimentally created, within the same animal, one optic nerve devoid of oligodendrocytes and their progenitors (the X-irradiated side) and one optic nerve containing the normal oligodendrocyte population (the untreated side). We now provide data demonstrating that 2-D gel protein profiles of normal and X-irradiated rat optic nerves can be compared to quickly isolate, from an in vivo model system, those proteins expressed by oligodendrocytes as well as those expressed by other cells which are regulated by oligendrocytes. The subsequent identification of these oligodendrocyte-associated proteins is accomplished by mass spectrometry. This experimental model system is especially powerful and unique since it retains all of the cell-cell interactions crucial to myelination. Taken together, these experiments should lead to a clearer understanding of the role of oligodendrocytes in CNS development and of the array of proteins associated with myelination.

描述（由申请人提供）：最近的研究检查了少突胶质细胞在发育中的作用，以及疾病或创伤后的作用，暴露了我们对少突胶质细胞功能的有限理解，并强调了建立新工具的必要性，这些工具将使我们能够揭示由少突胶质细胞调节的蛋白质的全部库并确定其功能。不幸的是，缺乏源自髓鞘少突胶质细胞的cDNA文库阻碍了鉴定源自这些细胞或受这些细胞调节的蛋白质的尝试。因此，我们建立了一个新的方法来阐明这样的蛋白质耦合技术的X射线照射，2-D凝胶电泳，质谱。具体来说，在以前的研究中，我们已经证明，少突胶质细胞可以选择性地消除从一个视神经的大鼠治疗的动物在出生时的X射线照射的单侧曝光。我们进一步表明，随着时间的推移，X射线照射的视神经成为重新填充与第二队列的少突胶质细胞从出生后第14天（P）on. This重新填充到X射线照射的神经细胞几乎恢复完整的补体少突胶质细胞通常发现在视神经P28。因此，利用这种方法，我们在同一动物中实验性地创建了一条没有少突胶质细胞及其祖细胞的视神经（X射线照射侧）和一条含有正常少突胶质细胞群体的视神经（未处理侧）。我们现在提供的数据表明，正常和X射线照射的大鼠视神经的2-D凝胶蛋白质谱可以进行比较，以快速分离，从体内模型系统中，少突胶质细胞表达的蛋白质，以及由其他细胞表达的那些由少突胶质细胞调节。这些少突胶质细胞相关蛋白的后续鉴定通过质谱法完成。这个实验模型系统特别强大和独特，因为它保留了对髓鞘形成至关重要的所有细胞-细胞相互作用。总之，这些实验应该导致更清楚地了解少突胶质细胞在中枢神经系统发育中的作用，以及与髓鞘形成相关的蛋白质阵列。