THE ROLE OF KERATOCAN IN CORNEAL BIOLOGY

描述（改编自申请人的摘要）： 本应用程序旨在更好地了解 角膜基质细胞特异性硫酸角质素蛋白聚糖（KSPG），角膜蛋白聚糖，在角膜 角膜蛋白聚糖在发育和维持 角膜稳态角膜不能正常发育 会导致角膜营养不良等疾病。Keratocan（Ktcn），Lumican (Lum)mimecan属于小亮氨酸蛋白聚糖（SLRP）基因 家人它们是脊椎动物角膜细胞外KSPG的主要成分 基质。已经表明角膜KSPGs调节胶原蛋白， 纤维形成，从而有助于角膜透明度。具体 目的1是通过基因靶向产生Ktcn-无效小鼠来检验假设 并检查Ktcn-/-角膜中的表型变化。老鼠Ktcn是 在角膜细胞中特异性表达。在胚胎中， 神经嵴细胞在角膜等组织的形态发生过程中迁移 间质、肢体和隔膜。我们假设角膜细胞谱系在角膜移植中起着重要的作用。 在角膜形态发生和维持角膜基质中的关键作用 功能具体目标2是阐明的分子基础 角膜细胞特异性基因表达和表征角膜细胞谱系。到 为了实现这一目标，研究人员已经表明，3.2 kb的Ktcn可以直接 外源基因（beta-geo）在成人角膜基质细胞中特异性表达 转基因小鼠（Tg）。目的2.1进一步明确角膜基质细胞特异性 通过一系列Ktcn启动子在3.2kb启动子内的顺式调节元件 使用转基因小鼠的缺失突变体。目的2.2是确认顺式元件 DNA酶I足迹法和电泳迁移率变动分析。的 Ktcnpr3.2-betageobpA Tg允许我们通过X-gal染色追踪角膜细胞。的 具体目的3是使用该Tg作为模型来研究 角膜基质细胞、结膜基质细胞和巩膜成纤维细胞 伤口愈合具体目的4是测试角膜细胞在角膜移植过程中的作用。 发育和上皮-粘液质相互作用中的遗传消融 四环素诱导的Ktcnpr3.1-rtTA/tetO-DT-A Tg模型中的角膜细胞。 目的4.1在角膜移植术中切除眼周神经嵴细胞 形态发生并检查对角膜和透镜的影响 形态发生目的4.2是消融成年动物的角膜细胞， 上皮和内皮对角膜细胞死亡的反应可以 阐明。

DESCRIPTION (adapted from the applicant's abstract): The long term objective of this application is to provide a better understanding of the role of keratocyte-specific keratan sulfate proteoglycan (KSPG), keratocan, in corneal function and the role of keratocan during development and in the maintenance of corneal homeostatis. Failure to have normal developmental process of the cornea will result in diseases such as corneal dystrophy. Keratocan (Ktcn), lumican (Lum), and mimecan belong to the small leucine-rich proteoglycan (SLRP) gene family. They are major components of extracellular KSPG in vertebrate corneal stroma. It has been suggested that corneal KSPGs modulate collagen fibrillogenesis and thus contribute to the corneal transparency. The specific aim 1 is to test the hypothesis by generating Ktcn-null mice via gene-targeting and examining the phenotypic changes in Ktcn-/- cornea. The mouse Ktcn is specifically expressed in keratocytes. In embryos, the Ktcn expression tracks the neural crest cells migrating during morphogenesis of tissue such as corneal stroma, limb and diaphragm. We hypothesize that keratocyte lineage plays a pivotal role in corneal morphogenesis and in the maintenance of corneal function. The specific aim 2 is to elucidate the molecular basis of keratocyte-specific gene expression and to characterize keratocyte lineage. To achieve this goal, the investigator has shown that the 3.2 kb Ktcn can direct a foreign gene (beta-geo) expression specifically to keratocytes in adult transgenic mice (Tg). Aim 2.1 is to further define the keratocyte-specific cis-regulatory element within the 3.2 kb promoter by a series of Ktcn promoter deletion mutants using transgenic mice. Aim 2.2 is to confirm the cis-element with DNase I footprinting and electrophoretic mobility shift assay. The Ktcnpr3.2-betageobpA Tg allow us to trace keratocytes via X-gal staining. The specific aim 3 is to use this Tg as a model to study cellular responses of corneal keratocytes, conjunctival keratocytes, and scleral fibroblasts during wound healing. The specific aim 4 is to test the role of keratocytes during development and in the epithelium-mysenchyme interactions by genetic ablation of keratocytes in a tetracycline inducible Ktcnpr3.1-rtTA/tetO-DT-A Tg model. Aim 4.1 is to ablate the periocular neural crest cells during corneal morphogenesis and to examine the consequences on corneal and lens morphogenesis. Aim 4.2 is to ablate the keratocytes in adult animals, thus epithelium and endothelium response to the keratocyte cell death can be elucidated.