BIOSYNTHESIS OF SCHWANN CELL EXTRACELLULAR MATRIX

施万细胞外基质的生物合成

• 批准号: 3403648
• 负责人: David J. Carey
• 金额: $ 17.77万
• 依托单位: GEISINGER CLINIC
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3403648
• 关键词: Schwann cells; affinity chromatography; antibody formation; axon; basement membrane; cell differentiation; collagen; complementary DNA; cytoskeleton; extracellular matrix; fibronectins; gel electrophoresis; heparan sulfate; immunocytochemistry; immunofluorescence technique; integrins; ion exchange chromatography; laboratory mouse; laboratory rabbit; laboratory rat; laminin; membrane activity; membrane proteins; messenger RNA; monoclonal antibody; mucopolysaccharides; myelination; neurochemistry; neurogenesis; nucleic acid sequence; oligonucleotides; peripheral nervous system; posttranslational modifications; protein biosynthesis; protein purification; protein sequence; proteoglycan; receptor binding; tissue /cell culture; western blottings

The long range goal of this project is to understand the role of extracellular matrix (ECM) in the development of the nervous system. The terminal differentiation of Schwann cells, which involves spreading on axonal membranes and myelin formation is strictly dependent upon contact with ECM. In vivo and in primary cell cultures this ECM is the basement membrane that is synthesized by the Schwann cells prior to and during the period of myelination. This proposal addresses 3 related aspects of this overall goal; namely, the structure, function and regulation of expression of cell surface heparan sulfate proteoglycans (HSPGs) of Schwann cells. Previous studies from our laboratory have shown that Schwann cells synthesize 2 distinct cell surface HSPGs that differ in their mode of association with the cell surface: a lipid anchored HSPG and an HSPG that is similar or identical to a previously identified epithelial transmembrane HSPG syndecan. Other experiments from our laboratory suggest that these HSPGs are involved in the interaction of Schwann cell with the basement membrane protein laminin. We propose to investigate by immunocytochemical methods the subcellular localization of these HSPGs in Schwann cells at various stages of differentiation in culture. We will also determine whether nerve cell or basement membrane contact, which are known to strongly influence Schwann cell behavior, affect the synthesis, distribution, or processing these HSPGs by Schwann cells. These experiments will utilize a combination of biochemical, immunological and molecular techniques. To examine the function of the cell surface HSPGs we will add specific antibodies to cultures of Schwann cells and nerve cells and observe their effects on the ability of the Schwann cells to assembly basement membrane de novo and to respond to exogenous basement membrane be spreading on and ensheathing and myelinating axons. We will also attempt to clone cDNA encoding the core protein of the lipid anchored Schwann cell HSPG. For these experiment a Schwann cell cDNA expression library will be screened with antibodies or oligonucleotides. Core protein sequences will be subcloned into the appropriate vectors for further analysis, such as DNA sequence determination.

本项目的长期目标是了解 细胞外基质（ECM）在神经系统发育中的作用。 的 许旺细胞的终末分化，包括在 轴突膜和髓鞘的形成严格依赖于接触 ECM的。 在体内和原代细胞培养物中，ECM是 膜是由许旺细胞合成之前和期间， 髓鞘形成期。 该提案涉及3个相关方面， 总体目标;即表达的结构、功能和调节 雪旺细胞表面硫酸乙酰肝素蛋白多糖（HSPGs）的表达。 我们实验室以前的研究表明， 合成2种不同细胞表面HSPG，它们的作用模式不同 与细胞表面结合的HSPG：脂质锚定的HSPG和 与先前鉴定的上皮跨膜细胞相似或相同 HSPG多配体蛋白聚糖 我们实验室的其他实验表明， HSPGs参与雪旺细胞与基底的相互作用 膜蛋白层粘连蛋白 我们建议通过免疫细胞化学 方法应用免疫组织化学方法，在体外培养的雪旺细胞中， 文化的不同阶段。 我们还将确定 无论是神经细胞还是基底膜接触， 强烈影响雪旺氏细胞行为，影响合成， 分布，或通过许旺细胞加工这些HSPG。 这些 实验将利用生物化学，免疫学和 分子技术 为了检测细胞表面HSPGs的功能，我们 将在雪旺氏细胞和神经细胞的培养物中加入特异性抗体 观察它们对雪旺细胞组装能力的影响 基底膜的新生和对外源性基底膜的反应， 在轴突上扩散、成鞘和髓鞘形成。 我们还将尝试 克隆编码脂质锚定雪旺细胞核心蛋白的cDNA HSPG 对于这些实验，将使用Schwann细胞cDNA表达文库。 用抗体或寡核苷酸筛选。 核心蛋白序列将 将其亚克隆到适当的载体中进行进一步分析，例如DNA 序列测定