Corneal Metalloproteinase-Matrix Interactions

角膜金属蛋白酶-基质相互作用

• 批准号: 7476255
• 负责人: Mark I Rosenblatt
• 金额: $ 22.49万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7476255
• 关键词: 3-Dimensional; Animals; Back; Biological Assay; Blindness; Breeding; Cell Line; Cells; Cicatrix; Cleaved cell; Collagen Type III; Collagen Type IV; Complement component C1s; Condition; Cornea; Corneal Injury; Data; Dependence; Deposition; Disease; Dose; End Point; Endopeptidases; Enzymes; Epithelial; Epithelial Cells; Epithelium; Extracellular Matrix; Gel; Gene Activation; Gene Expression; Genes; Goals; Green Fluorescent Proteins; Healed; Immunohistochemistry; In Vitro; Incubated; Injury; Investigation; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Laser injury; Lasers; Localized; Mass Spectrum Analysis; Matrilysin; Matrix Metalloproteinases; Measurement; Measures; Messenger RNA; Migration Assay; Mus; N-terminal; Organ; Pattern; Peptide Hydrolases; Polymerase Chain Reaction; Preparation; Production; Proteins; Range; Regulation; Role; Site; Substrate Specificity; System; Techniques; Tenascin; Time; Tissues; Transcript; Transcriptional Activation; Transgenic Animals; Transgenic Mice; Translating; Western Blotting; Wild Type Mouse; Wound Healing; base; corneal epithelium; corneal scar; decorin; enzyme activity; healing; in vitro Assay; insight; macromolecule; migration; prevent; promatrilysin; promoter; protein expression; research study; two-dimensional; wound

DESCRIPTION (provided by applicant): Loss of corneal clarity is a common endpoint of many blinding conditions. Corneal opacification is a major cause of blindness worldwide. Our laboratory is studying the mechanisms associated with corneal wound repair that regulate coneal clarity. Specifically, we have identified an endogenous matrix metalloproteinase within the cornea which helps prevent corneal opcification after an excimer laser wound. Matrilysin (MMP-7) deficient mice have exuberant scarring following laser injuries which are innocuous to their wild-type counterparts. In this application we will investigate the interaction of MMP-7 with extracelluar matrix (ECM) components localized to native and wounded corneas. Specific Aim A will use a combination of immunohistochemistry, western blotting, zymography, and transgenic animals to localize the sites and quantify the amount of MMP-7 expressed in wounded and unwounded corneas. Specific Aim B extends these studies to determine the matrix substrate specificities of corneal epithelium- and keratocyte-derived MMP-7 in vitro. In this aim we will examine corneal celI-ECM interactions by assaying the migration of epithelium on 2-dimensional matrix gels, and the proliferation of keratocytes in 3-dimensional matrix gels. Specific Aim C will translate the in vitro results to MMP-7 knockout mice. The expression of xtracellular matrix components in wild-type and matrilysin knockout mice after excimer laser wounding will be evaluated by immunohistochemistry, western blotting and real-time PCR. The second portion of Aim C will carefully examine the MMP-7 dependent breakdown products of corneal extracellular matrix in these mice. Lastly, using MMP-7/ECM double knockout mice, the requirement of specific extracellular matrix components for reduced corneal clarity will be investigated. Our studies of protease-matrix interactions in wounded corneas will further our understanding of the mechanisms for maintaining corneal clarity. Results of these studies in the cornea may ultimately be helpful in understanding healing in other organs as well.

描述（由申请人提供）：角膜透明度丧失是许多致盲情况的常见终点。角膜混浊是全世界失明的主要原因。 我们的实验室正在研究与调节角膜透明度的角膜伤口修复相关的机制。 具体来说，我们在角膜内发现了一种内源性基质金属蛋白酶，它有助于防止准分子激光损伤后的角膜混浊。 Matrilysin (MMP-7) 缺陷小鼠在激光损伤后会出现大量疤痕，而这对于野生型小鼠来说是无害的。 在此应用中，我们将研究 MMP-7 与本地角膜和受伤角膜的细胞外基质 (ECM) 成分的相互作用。 具体目标 A 将结合免疫组织化学、蛋白质印迹、酶谱分析和转基因动物来定位位点并量化受伤和未受伤角膜中表达的 MMP-7 量。 具体目标 B 扩展了这些研究，以确定体外角膜上皮和角膜细胞衍生的 MMP-7 的基质底物特异性。 在此目的中，我们将通过测定二维基质凝胶上上皮的迁移和三维基质凝胶中角膜细胞的增殖来检查角膜细胞-ECM相互作用。 具体的 Aim C 将体外结果转化为 MMP-7 敲除小鼠。将通过免疫组织化学、蛋白质印迹和实时PCR评估准分子激光损伤后野生型和基质溶素敲除小鼠中细胞外基质成分的表达。 Aim C 的第二部分将仔细检查这些小鼠角膜细胞外基质的 MMP-7 依赖性分解产物。 最后，使用 MMP-7/ECM 双敲除小鼠，将研究降低角膜透明度所需的特定细胞外基质成分。 我们对受伤角膜中蛋白酶-基质相互作用的研究将进一步加深我们对维持角膜清晰度的机制的理解。 这些角膜研究的结果最终可能有助于了解其他器官的愈合情况。