NEUROBIOLOGY OF DEVELOPMENT

发育的神经生物学

• 批准号: 3100085
• 负责人: WILLIAM T NORTON
• 金额: $ 74.59万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3100085
• 关键词: developmental neurobiology

This program contains four projects devoted to the neurochemistry and cell biology of nervous system development. All studies are to be performed in rats. We propose: (1) to isolate and purify glial precursor cells from the developing and mature rat brain, to follow the differentiation of these cells in culture, and to test the hypothesis that these precursor cells serve as a reservoir for proliferating astrocytes and oligodendrocytes following injury. (2) To extend our studies of cellular localization of the glial-specific enzymes: Yp and Yb isoforms of glutathione-stransferase, carbonic anhydrase, glutamine synthetase and the RLM6 isozyme of cytochrome P-450 in the developing normal and myelin-deficient rat. These studies which will include immunostaining of tissue sections and cell cultures and enzyme assays of bulk isolated cells, are expected to help resolve some current issues in glial cell development, with particular emphasis on protoplasmic astrocytes. (3) To study the expression of a novel 66KD CNS-specific neurofilament protein in rat cerebellum during development and in cultured granule cells. The axonal cytoskeleton of the granule cells will be examined by electron microscopy and immuno-EM in vivo and in vitro. A cDNA probe will be constructed to the 66 KD NF protein and used for in situ hybridization in tissues and in Northern blot analyses of extracted RNAs. (4) To examine the heat-shock (stress) response in the CNS of Lewis rats induced to develop EAE, a disease involving inflammation, demyelination and extensive reactive gliosis; and in glial cells in culture during development and differentiation. Heat-stress mRNA and protein will be analyzed by Northern and Western blots, in situ hybridization, immuno-cytochemistry and in vitro protein synthesis. The methodology for these four studies includes cell isolation, cell culture, immunocytochemistry, enzyme assays, cDNA construction, gene sequencing, RNA isolation, Northern blots, Western blots, protein synthesis, gel electrophoresis, in situ hybridization, subcellular fractionation and polysome analysis. The long range goals of the program are to provide detailed information on some well defined processes of development and differentiation of the nervous system, so that we may better understand how they are perturbed by genetic or environmental influences. The results obtained should also lead to information concerning the generation of reactive astrocytes following injury and of oligodendro- cytes for remyelination.

该计划包含四个致力于神经化学和 神经系统发育的细胞生物学。 所有的学习都要 在大鼠中进行。我们建议：（1）分离纯化胶质前体 来自发育和成熟大鼠大脑的细胞，遵循 这些细胞在培养物中分化，并检验假设 这些前体细胞充当增殖的储存库 损伤后的星形胶质细胞和少突胶质细胞。 (2) 扩展我们的 神经胶质特异性酶 Yp 和 Yb 的细胞定位研究 谷胱甘肽转移酶、碳酸酐酶、谷氨酰胺同工型 发育中细胞色素 P-450 的合成酶和 RLM6 同工酶 正常和髓磷脂缺乏的大鼠。 这些研究将包括 组织切片和细胞培养物的免疫染色以及酶测定 批量隔离电池有望帮助解决当前的一些问题 胶质细胞发育，特别强调原生质 星形胶质细胞。 (3) 研究新型66KD CNS特异性的表达 大鼠小脑发育过程中的神经丝蛋白 培养的颗粒细胞。 颗粒细胞的轴突细胞骨架 将通过电子显微镜和免疫电镜在体内和体内进行检查 体外。 将针对 66 KD NF 蛋白构建 cDNA 探针并使用 用于组织中的原位杂交和 Northern blot 分析 提取的 RNA。 (4) 检验热休克（应激）反应 Lewis 大鼠的中枢神经系统诱发 EAE，这是一种涉及 炎症、脱髓鞘和广泛的反应性神经胶质增生；和神经胶质 发育和分化过程中培养的细胞。热应激mRNA 蛋白质将通过 Northern 和 Western 印迹进行原位分析 杂交、免疫细胞化学和体外蛋白质合成。这 这四项研究的方法包括细胞分离、细胞培养、 免疫细胞化学、酶测定、cDNA 构建、基因测序、 RNA 分离、Northern 印迹、蛋白质印迹、蛋白质合成、凝胶 电泳、原位杂交、亚细胞分级分离和 多核糖体分析。该计划的长期目标是提供 有关一些明确定义的开发过程的详细信息 神经系统的分化，让我们更好地了解 他们如何受到遗传或环境影响的困扰。这 获得的结果还应导致有关 损伤后产生反应性星形胶质细胞和少突胶质细胞 用于髓鞘再生的细胞。