Molecular Aspects of Corneal Epithelial Migration

角膜上皮迁移的分子方面

• 批准号: 7880422
• 负责人: Mary Ann Stepp
• 金额: $ 28.81万
• 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 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7880422
• 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; Environment; 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; Mitomycins; Modeling; Molecular; Mus; Nose; Null Lymphocytes; Organ Culture Techniques; Patients; Peripheral; Phenotype; Play; Protein Family; Proteins; 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

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 CESCDare 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. Reducingthe frequency of erosions by inhibiting the activity of MMPs and ADAM17 using the small molecule inhibitor GM6001. 2. To test the hypothesis that CESCDis caused by the induction of proliferationof early transit amplfying (eTA)cells during reepithelialization, CESCDwill 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 CESCDby increasing the size of the wounds above 1.5mm and measuring eTA cellproliferation. 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 CESCDby 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-mediatedsignaling are responsible for the increasein keratinocyte migration that occurs in response to TGFpl and compare to corneas in organ culture by: 3a. Determining if migration and EGFreceptor signaling differs between wt and syndl null cellsin 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 EGFinduce 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.

作为对角膜创伤的反应，在伤口最初发生后，可能会出现自发的复发侵蚀。 痊愈了。累及周边角膜的伤口靠近角膜上皮干细胞，可导致角膜 上皮干细胞缺乏症(CESCD)，导致眼睛瘙痒和血管生长到角膜上 并干扰视力。在老化的角膜中，由于以下疾病，伤口的愈合变得缓慢 糖尿病。利用不同品系的正常和转基因小鼠及小鼠上皮细胞的培养 细胞，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。过去的研究导致了一种假设，即被称为整合素的蛋白质的表达发生了变化 当愈合延迟时，以及当出现反复侵蚀和/或CESCD时。为了更好地理解 针对这些情况的原因和开发治疗方法，提出了以下具体目标：1.在 在小鼠体内创伤性反复侵蚀是否是由于粘连的不完全重组所致 这些复合体进而通过：LA诱导ADAM17的慢性激活。黏附复合体的评价 浅层角膜切除术后小鼠全角膜三维共聚焦成像和透射电子显微镜重建 清创伤口，伊布。角膜术后ADAM-17上调和激活时机的确定 清创伤人，还有IC。通过抑制MMPs和MMPs的活性来减少侵蚀的频率 ADAM17使用小分子抑制剂GM6001。2.检验CESCD是由 在再上皮化过程中诱导早期传递放大(ETA)细胞的增殖，CESCD将被 整体共聚焦测量角膜上K8+Muc5ac+杯状细胞的体内定量 术后4wk显微镜观察手动角膜清创伤模型的变化情况：2a。 将创面直径增大至1.5 mm以上，检测ETA细胞增殖情况，诱导CESCD。 2B，产生环形伤口，留下中央角膜上皮细胞岛，以减少增殖和 损伤后ETA和CESCs的迁移和2c.通过使用以下两种药物之一治疗鼻缘来减少CESCD 在清创创面后立即使用丝裂霉素C或丝裂霉素AG1478，这通常会导致CESCD. 为了检测整合素表达的改变和EGFR介导的信号转导是否导致了这种增加 角质形成细胞在TGFpl作用下的迁移，与器官培养中的角膜相比：3A。 确定wt和syndl空细胞对 TGFp1和/或EGF，3b。在wt小鼠皮肤细胞和人角膜中诱导syndl零表型 用Synd1导向的siRNA表达慢病毒3c感染上皮细胞系 在器官培养中损伤wt和syndl空小鼠角膜以确定转化生长因子(31)和表皮生长因子(EGF)是否诱导 相似的反应。在了解角膜如何愈合方面取得了重大进展，但有所改善 需要为因创伤而病理性愈合的患者开发治疗方案。