FIBROBLAST DIFFERENTIATION DURING EYE DEVELOPMENT

眼睛发育过程中的成纤维细胞分化

• 批准号: 3255627
• 负责人: GARY W CONRAD
• 金额: $ 16.63万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3255627
• 关键词: binding proteins; cell differentiation; chemical hydration; chick embryo; chromatography; cornea; corneal stroma; embryogenesis; extracellular matrix; eye; fibroblasts; histogenesis; innervation; keratan sulfate; laboratory mouse; laboratory rabbit; monoclonal antibody; neural crest; nonmammalian vertebrate embryology; organ culture; peptide chemical synthesis; protein sequence; proteoglycan; radioassay; spectrometry; wound healing

The most distinctive product of neural crest differentiation in the cornea is keratan sulfate proteoglycan (KSPG). This extracellular, sulfated, N-linked glycoprotein is more abundant in cornea than in any other tissue. Although good chemical characterization of KS polysaccharide has been performed, little is known about the core proteins of KSPG. Some studies suggest multiple forms of corneal KSPG. Our preliminary data suggest as many as 5 proteins associated covalently or non-covalently with KSPG. Synthesis of KS polysaccharide by isolated, differentiated corneal stroma cells (keratocytes) in vitro occurs for only short periods of time. During corneal wound healing, synthesis of antigenically altered KS in the scar is detected. During normal corneal development in avian embryos, neural crest cells do not express antigens of mature KS until after the cells invade the primary corneal stromal. Corneal nerves invade the stroma shortly after extracellular KS is detected. Given this background, we propose to test three theories. Hypothesis 1: There are two classes of KSPG: a "constitutive" form present in most tissues including cornea, and a "tissue-specific" form; KSPG-binding proteins are associated with both forms. Chick corneal KSPG core proteins will be isolated, 10- 20 residues of each N-terminal sequence will be determined, peptides duplicating these sequences will be synthesized chemically, polyclonal antibodies will be prepared against each peptide, and expression of the antigens will be localized in developing chick eyes and other tissues. Hypothesis 2: Cornea- specific KSPG synthesis is regulated by the extracellular environment: 1) diffusible factors from other corneal cell-types or aqueous humor, 2) insoluble components of the stromal extracellular matrix, or 3) physical forces and/or dehydration of the stroma. Antibodies will be used to determine whether or when KSPG core protein antigens stop being expressed in vitro. One organ culture system for neural crest differentiation will allow experimental alteration of corneal cell-types and perturbation of extracellular matrix. Another organ culture system will assay stability of keratocyte differentiation within altered matrices and test the effect of constant tensile stress on KSPG synthesis. Hypothesis 3: Nerves invade the corneal stroma during embryogenesis in response to extracellular deposition of KSPG in the stroma. Inhibitors of N-linked glycosylation and processing will be used to perturb synthesis of KSPG, and effects on corneal innervation will be recorded.

神经波峰分化的最独特的产物 角膜是硫酸角酸盐蛋白聚糖（KSPG）。 这个细胞外， 硫化，N连接的糖蛋白在角膜中比在角膜中更丰富 任何其他组织。 虽然KS的良好化学表征 多糖已经执行，对核心知之甚少 KSPG的蛋白质。 一些研究表明多种形式的角膜 kspg。 我们的初步数据表明多达5种蛋白质 与KSPG共价或非共价关联​​。 合成 通过分离的，分化的角膜基质细胞的KS多糖 （角膜细胞）体外仅发生短时间。 在角膜伤口愈合期间，抗原改变的合成 检测到疤痕中的KS。 在正常角膜发育期间 鸟类胚胎，神经rest细胞不表达成熟的抗原 KS直到细胞侵入主要角膜基质后。 角膜神经在细胞外KS后不久侵入基质 检测到。 鉴于此背景，我们建议测试三个 理论。 假设1：有两类KSPG： 大多数组织中存在的“本构”形式，包括角膜和 “组织特异性”形式； KSPG结合蛋白与 两种形式。 将分离出小鸡角膜KSPG核心蛋白，10- 将确定每个N末端序列的20个残基， 复制这些序列的肽将合成 化学上，将准备多克隆抗体 肽和抗原的表达将定位在 发展雏鸡的眼睛和其他组织。 假设2：角膜 - 特定的KSPG合成受细胞外调节 环境：1）来自其他角膜细胞类型的可扩散因子 或水性幽默，2）基质的不溶性成分 细胞外基质或3）物理力和/或脱水 基质。 抗体将用于确定是否或何时 KSPG核心蛋白抗原停止在体外表达。 一 神经rest分化的器官培养系统将允许 角膜细胞类型和扰动的实验改变 细胞外基质。 另一个器官文化系统将分析 改变矩阵和角膜细胞分化的稳定性 测试恒定拉伸应力对KSPG合成的影响。 假设3：神经在 响应KSPG细胞外沉积的胚胎发生 基质。 N连接糖基化和加工的抑制剂 将用于扰动KSPG的合成和对角膜的影响 神经将记录。