FIBROBLAST DIFFERENTIATION DURING EYE DEVELOPMENT

最具特色的神经脊分化产物 角膜是硫酸角蛋白多糖(KSPG)。这种细胞外的， 硫酸盐化的N-连接糖蛋白在角膜中比在 任何其他组织。尽管KS具有良好的化学特性 已经进行了多糖的研究，对其核心知之甚少 KSPG蛋白。一些研究表明有多种形式的角膜 KSPG。我们的初步数据显示有多达5种蛋白质 与KSPG共价或非共价结合。一种新的合成方法 分离分化的角膜基质细胞提取KS多糖的研究 (角质形成细胞)在体外只会发生很短的时间。 在角膜伤口愈合过程中，抗原性改变的合成 检测到疤痕中的KS。在正常的角膜发育过程中 禽类胚胎、神经脊细胞不表达成熟抗原 KS直到细胞侵入原发角膜基质之后。 角膜神经在细胞外KS后不久侵入基质 检测到。鉴于这一背景，我们建议测试三个 理论。假设1：有两类KSPG：A 在包括角膜在内的大多数组织中都存在“构成”形式，以及 一种“组织特异性”形式；KSPG结合蛋白与 两种形式都有。将分离鸡角膜KSPG核心蛋白，10- 将确定每个N-末端序列的20个残基， 复制这些序列的多肽将被合成 从化学上讲，将针对每一种情况制备多克隆抗体 多肽，抗原的表达将定位于 发育小鸡的眼睛和其他组织。假设2：角膜-- 特异性KSPG的合成受细胞外调节 环境：1)来自其他角膜细胞类型的扩散因素 或房水，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。抗体将被用来确定是否或何时 KSPG核心蛋白抗原在体外不再表达。一 用于神经脊分化的器官培养系统将允许 实验性角膜细胞类型的改变和微扰 细胞外基质。另一种器官培养系统将检测 改变的基质和基质中角膜细胞分化的稳定性 测试恒定张应力对KSPG合成的影响。 假设3：神经侵入角膜基质 KSPG胞外沉积对胚胎发生的影响 基质。N-连接糖基化的抑制剂及其加工 会被用来干扰KSPG的合成，并对角膜产生影响 神经支配将被记录下来。

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.