Expressions of Specialized Collagens in the Cornea

角膜使用细胞外基质(ECM)分子，特别是胶原蛋白，来建立一个 具有两个重要功能的建筑：折射和光的传输。我们的总体目标是 了解角膜如何调节一组特定的 胶原蛋白实现了这一架构。角膜基质中的胶原纤维细小，直径均匀。 结构，以离散的距离均匀分布，并排列在正交层中。我们假设 纤维状胶原XXIV和面胶原XIV有助于调节纤维直径，而且 Facit胶原XII调节角膜基质中纤维的间距。 为了解决这些假设，我们在目标1中提出了确定角膜发生了什么变化 一种分泌减少数量的XII型胶原的转基因小鼠。我们的初步数据显示 在转基因角膜中，胶原纤维间隔更紧密。原纤维直径、原纤维间距 并将测量转基因小鼠角膜的生物力学强度，并与野生型小鼠进行比较。 我们还将评估其他基质分子如何受到XII胶原蛋白缺乏的影响，以及 使角膜中的XII胶原蛋白水平降低的眼科学要求。有可能这些都是 老鼠代表了人类角膜疾病圆锥角膜的动物模型。在目标2中，我们将确定 XII胶原蛋白是否是角膜中的蛋白多糖，以及类似于其在软骨中的结构的胶原蛋白。 这在角膜中很有意义，因为蛋白多糖与正确的胶原纤维有关 间隔，以及已知的角膜蛋白多糖基因被敲除的小鼠没有表现出显著的 角膜纤维间距的改变。如果XII胶原蛋白是一种蛋白多糖，我们将测量 它所代表的总蛋白多糖。在目标3中，我们将演示新发现的纤维状胶原类型 XXIV和Facit胶原XIV是发育中的鸡角膜中纤维直径的调节因子。金额的多少 角膜中分泌的XXIV和XIV胶原蛋白将通过注射到角膜中而减少 编码每种胶原蛋白的截短版本。截断的链和正常的链将合并并被降解 细胞内，减少XIV和XXIV胶原分泌量。原纤体建筑的变化 由显性负性表型引起的结果将通过透射电子显微镜进行评估。

The cornea uses extracellular matrix (ECM) molecules, in particular, collagens, to set up an architecture that serves two crucial functions: refraction and transmission of light. Our overall objective is to understand how the cornea regulates the synthesis, assembly, and differential expression of a specific set of collagens to achieve this architecture. Collagen fibrils in the corneal stroma are thin, uniform diameter structures, evenly spaced apart at discreet distances, and arranged in orthogonal layers. We hypothesize that fibrillar collagen XXIV and FACIT collagen XIV contribute to regulating the fibril diameters, and that FACIT collagen XII regulates the spacing of the fibrils in the corneal stroma. To address these hypotheses we propose in Aim 1 to determine what changes occur in the cornea of a transgenic mouse that secretes reduced quantites of type XII collagen. Our preliminary data indicate that collagen fibrils are spaced much closer together in the transgenic cornea. The fibril diameters, fibril spacing and biomechanical strength of transgenic mouse corneas will be measured and compared to wild type mice. We will also assess how other matrix molecules are affected by a paucity of collagen XII and what are the ophthalmologic consquences of making a cornea with reduced levels of collagen XII. It is possible that these mice represent an animal model for the human corneal disease, keratoconus. In Aim 2, we will determine whether collagen XII is a proteoglycan in the cornea, as well as a collagen, similar to its structure in cartilage. This is of interest in the cornea because proteoglycans have been implicated in correct collagen fibril spacing, and mice with the known corneal proteoglycan genes knocked out have not shown significant alterations in corneal fibril spacing. If collagen XII is a proteoglycan, we will measure what percentage of the total proteoglycan it represents. In Aim 3 we will demonstrate whether newly identified fibrillar collagen type XXIV and FACIT collagen XIV are fibril diameter regulators in the developing chick cornea. The amount of collagen XXIV and XIV secreted in the cornea will be reduced by injecting into the cornea in ovo virus encoding truncated versions of each collagen. Truncated and normal chains will combine and be degraded intracellularly, reducing the amount of secreted collagen XIV and XXIV. The alterations in fibril architecture resulting from the dominant negative phenotype will be evaluated by transmission electron microscopy.