Molecular Aspects of Corneal Epithelial Migration

角膜上皮迁移的分子方面

• 批准号: 7643853
• 负责人: Mary Ann Stepp
• 金额: $ 38.88万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7643853
• 关键词: 3-Dimensional; Adhesions; Affect; Aging; Anatomy; Animal Model; BALB/cJ Mouse; Biological Models; Birth; Bromodeoxyuridine; Cell Culture Techniques; Cell Line; Cell Proliferation; Cells; Chronic; Complex; Confocal Microscopy; Cornea; Corneal Injury; Debridement; Diabetes Mellitus; Disease; EGF gene; Environment; Epidermal Growth Factor Receptor; Epithelial Cells; Epithelium; Etiology; Excision; Experimental Models; Exposure to; Eye; Frequencies; Funding; Goblet Cells; Healed; Hemidesmosomes; Human; Image; Impaired wound healing; Inbred BALB C Mice; Injury; Integrins; Island; Knockout Mice; Lead; Left; MMP3 gene; MMP9 gene; Manuals; Matrix Metalloproteinases; Measures; Mediating; Mitomycins; Modeling; Molecular; Mus; Nose; Null Lymphocytes; Organ Culture Techniques; Patients; Peripheral; Phenotype; Play; Protein Family; Proteins; Receptor Signaling; Recurrence; Research; Role; Severities; Signal Transduction; Site; Skin; Small Interfering RNA; Subfamily lentivirinae; Surface; TNF-alpha converting enzyme; Testing; Time; Tissues; Transmission Electron Microscopy; Trauma; Up-Regulation; Variant; Vision; Work; Wound Healing; corneal epithelial stem cells; corneal epithelium; cytokine; eye blood vessel; healing; improved; in vivo; inhibitor/antagonist; keratinocyte; limbal; migration; mouse model; ocular surface; protein expression; response; small molecule; syndecan; wound

DESCRIPTION (provided by applicant): In response to trauma to the cornea, spontaneous recurrent erosions can develop after wounds have initially healed. Wounds that involve the peripheral cornea near the corneal epithelial stem cells can lead to a corneal epithelial stem cell deficiency (CESCD) that causes scratchy eyes and blood vessels to grow onto the cornea and interfere with vision. In the aging cornea, healing of wounds becomes slower due to diseases such as diabetes. Using various strains of normal and genetically modified mice and the culture of mouse epithelial cells, experimental model systems have been developed to study these conditions and to develop new treatments. Past research has lead to the hypothesis that expression of proteins called integrins is altered when healing is delayed and when recurrent erosions and/or CESCD are present. To better understand the causes and develop treatments for these conditions, the following Specific Aims are proposed: 1. To test in vivo in mice whether trauma-induced recurrent erosions are due to incomplete reassembly of adhesion complexes which, in turn, induces chronic activation of ADAM17 by: la. Evaluating adhesion complex reassembly using 3-D confocal imaging of whole mouse corneas and TEM after superficial keratectomy and debridement wounds, Ib. Determining the timing of ADAM-17 up-regulation and activation after corneal debridement wounding, and Ic. Reducing the frequency of erosions by inhibiting the activity of MMPs and ADAM17 using the small molecule inhibitor GM6001. 2. To test the hypothesis that CESCD is caused by the induction of proliferation of early transit amplfying (eTA) cells during reepithelialization, CESCD will be quantified in vivo by measuring K8+Muc5ac+ goblet cells on the cornea using whole mount confocal microscopy 4 wk after the following variations of the manual corneal debridement wound model: 2a. Inducing CESCD by increasing the size of the wounds above 1.5 mm and measuring eTA cell proliferation. 2b, Generating annular wounds leaving a central corneal epithelial cell island to reduce proliferation and migration of eTA and CESCs after injury, and 2c. Reducing CESCD by treating the nasal limbus with either mitomycin C or tryphostin AG1478 immediately after debridement wounds that normally induce CESCD. 3. To test whether altered integrin expression and EGFR-mediated signaling are responsible for the increase in keratinocyte migration that occurs in response to TGFpl and compare to corneas in organ culture by: 3a. Determining if migration and EGF receptor signaling differs between wt and syndl null cells in response to TGFpl and/or EGF, 3b. Inducing the syndl null phenotype in wt mouse skin cells and in a human corneal epithelial cell line by infecting cells with syndl directed siRNA-expressing lentivirus, 3c. Using debridement wounded wt and syndl null mouse corneas in organ culture to determine whether TGF(31 and EGF induce similar responses. Significant progress has been made in understanding how the cornea heals but improved treatment options need to be developed for patients with pathological healing due to trauma.

描述（由申请人提供）：作为对角膜创伤的反应，在伤口最初愈合后可发生自发性复发性糜烂。涉及角膜上皮干细胞附近的外周角膜的伤口可导致角膜上皮干细胞缺陷（CESCD），其导致眼睛发痒和血管生长到角膜上并干扰视力。在老化的角膜中，由于糖尿病等疾病，伤口愈合变得缓慢。使用各种品系的正常小鼠和转基因小鼠以及小鼠上皮细胞的培养物，已经开发了实验模型系统来研究这些条件并开发新的治疗方法。过去的研究已经导致了这样的假设，即当愈合延迟和复发性糜烂和/或CESCD存在时，称为整合素的蛋白质的表达会改变。为了更好地了解这些疾病的原因并开发治疗方法，提出了以下具体目标：1。为了在小鼠体内测试创伤诱导的复发性糜烂是否是由于粘附复合物的不完全重新组装，粘附复合物进而通过以下方式诱导ADAM 17的慢性激活：在浅层角膜切除术和清创伤口后，使用整个小鼠角膜的3-D共聚焦成像和TEM评价粘附复合物的重新组装，Ib。确定角膜清创创伤后ADAM-17上调和激活的时间，和Ic.通过使用小分子抑制剂GM 6001抑制MMPs和ADAM 17的活性来减少糜烂的频率。2.为了检验CESCD是由上皮再形成过程中早期转运扩增（eTA）细胞增殖的诱导引起的假设，将在以下手动角膜清创伤口模型的变化后4周，通过使用整体封片共聚焦显微镜测量角膜上的K8+ Muc 5ac+杯状细胞来体内定量CESCD：2a.通过将伤口的大小增加到1.5 mm以上并测量eTA细胞增殖来诱导CESCD。2b，产生环形伤口，留下中央角膜上皮细胞岛以减少损伤后eTA和CESC的增殖和迁移，和2c。在通常诱导CESCD的清创伤口后立即用丝裂霉素C或Tryphostin AG 1478处理鼻利姆布斯，减少CESCD。3.为了测试改变的整联蛋白表达和EGFR介导的信号传导是否负责响应于TGF β 1而发生的角质形成细胞迁移的增加，并通过以下方式与器官培养物中的角膜进行比较：3a.确定响应于TGF β 1和/或EGF的wt和syndl无效细胞之间的迁移和EGF受体信号传导是否不同，3b。通过用syndl定向的siRNA表达慢病毒感染细胞，在野生型小鼠皮肤细胞和人角膜上皮细胞系中诱导syndl无效表型，3c.在器官培养中使用清创损伤的wt和syndl缺失的小鼠角膜来确定TGF β 1和EGF是否诱导类似的反应。在理解角膜如何愈合方面已经取得了重大进展，但需要为因创伤而病理性愈合的患者开发改进的治疗方案。