Expressions of Specialized Collagens in the Cornea

描述：角膜使用细胞外基质（ECM）分子，特别是胶原，来建立一个具有两个关键功能的结构：光的折射和透射。我们的总体目标是了解角膜如何调节一组特定胶原蛋白的合成、组装和差异表达，以实现这种结构。角膜基质中的胶原原纤维是薄的、直径均匀的结构，在离散的距离上均匀间隔，排列成正交层。我们假设纤维胶原XXIV和FACIT胶原XIV有助于调节原纤维直径，FACIT胶原XII调节角膜基质中原纤维的间距。为了解决这些假设，我们在目标1中提出，以确定在分泌减少的XII型胶原蛋白的转基因小鼠的角膜中发生了什么变化。我们的初步数据表明，胶原原纤维在转基因角膜中间距更近。测定转基因小鼠角膜的原纤维直径、原纤维间距和生物力学强度，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胶原缺乏的影响，以及制造具有低水平XII胶原的角膜的眼科后果。这些小鼠可能代表了人类角膜疾病圆锥角膜的动物模型。在Aim 2中，我们将确定XII胶原是否是角膜中的蛋白多糖，以及与软骨结构相似的胶原蛋白。这在角膜中很有趣，因为蛋白多糖与正确的胶原纤维间距有关，而敲除已知角膜蛋白多糖基因的小鼠角膜纤维间距没有显着改变。如果十二型胶原蛋白是一种蛋白多糖，我们将测量它占总蛋白多糖的百分比。在Aim 3中，我们将证明新发现的纤维胶原型XXIV和FACIT胶原型XIV是否在发育中的鸡角膜中是纤维直径调节剂。通过将编码每种胶原蛋白截断版本的卵形病毒注射到角膜中，可以减少角膜中分泌的胶原蛋白XXIV和XIV的数量。截断的和正常的链将结合并在细胞内降解，减少胶原蛋白XIV和XXIV的分泌量。由显性阴性表型引起的纤维结构改变将通过透射电子显微镜进行评估。

DESCRIPTION: 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 quantities 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 consequences 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.