THE ROLE OF KERATOCAN IN CORNEAL BIOLOGY

描述(改编自申请人的摘要)：长期目标 此应用程序旨在更好地了解 角膜中的角质细胞特异性硫酸角蛋白多糖(KSPG) 角质形成蛋白在发育和维持中的功能和作用 角膜同种异体。没有正常的角膜发育过程 会导致角膜营养不良等疾病。Keratocan(Ktcn)，LUMICAN (Lum)和Mimecan属于富含亮氨酸的蛋白多糖(SLRP)基因 一家人。它们是脊椎动物角膜细胞外KSPG的主要成分 斯特尔玛。已有研究表明，角膜KSPG对胶原的调节作用 纤维形成，因此有助于角膜的透明度。具体的 目标1是通过基因打靶产生Ktcn缺失的小鼠来检验这一假说 并检测Ktcn-/-角膜的表型变化。鼠标Ktcn是 在角质形成细胞中特异表达。在胚胎中，Ktcn的表达跟踪 角膜等组织形态发生过程中神经脊细胞的迁移 间质、肢体和横隔膜。我们假设角质形成细胞系在 角膜形态发生和角膜维持中的关键作用 功能。具体目的2是为了阐明 角质细胞特异性基因的表达和角质细胞谱系的特征。至 为了实现这一目标，研究人员已经表明，3.2kb的Ktcn可以指导 外源基因(β-geo)在成人角质形成细胞中的特异性表达 转基因小鼠(TG)。目标2.1是进一步定义特定于角膜细胞的 一系列Ktcn启动子在3.2kb启动子中的顺式调控元件 使用转基因小鼠的缺失突变体。目标2.2是确认顺式元件 用DNase I足迹和凝胶迁移率改变分析。这个 Ktcnpr3.2-betageobpA TG允许我们通过X-Gal染色来追踪角质形成细胞。这个 具体目标3是使用这个TG作为模型来研究细胞反应 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是测试角质形成细胞在 遗传消融在上皮细胞-肌丝体相互作用中的发育 四环素诱导的Ktcnpr3.1-RTTA/Teto-dt-A TG模型中角膜基质细胞的表达。 目标4.1是在角膜手术中消融眼周神经脊细胞 形态发生及对角膜和晶状体的影响 形态发生。目的4.2是为了去除成年动物的角膜基质细胞，从而 上皮细胞和内皮细胞对角质形成细胞死亡的反应可 已澄清。

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.