Corneal Metalloproteinase-Matrix Interactions

角膜金属蛋白酶-基质相互作用

• 批准号: 7289246
• 负责人: Mark I Rosenblatt
• 金额: $ 15.5万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 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 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7289246
• 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（由申请方提供）：角膜透明度丧失是许多设盲条件的常见终点。角膜混浊是世界范围内致盲的主要原因。 我们的实验室正在研究与角膜创伤修复相关的调节角膜透明度的机制。 具体来说，我们已经确定了角膜内的内源性基质金属蛋白酶，这有助于防止准分子激光创伤后角膜混浊。 基质溶解素（MMP-7）缺陷小鼠在激光损伤后有丰富的瘢痕形成，这对野生型小鼠无害。 在本申请中，我们将研究MMP-7与细胞外基质（ECM）成分的相互作用，定位于天然和受伤的角膜。 具体目标A将使用免疫组织化学、蛋白质印迹、酶谱和转基因动物的组合来定位受伤和未受伤角膜中表达的MMP-7的位点并定量。 特定目的B扩展了这些研究，以确定角膜上皮和角膜基质细胞衍生的MMP-7的体外基质底物特异性。 在这个目标中，我们将通过测定上皮细胞在二维基质凝胶上的迁移和角膜细胞在三维基质凝胶中的增殖来研究角膜细胞-ECM的相互作用。 Specific 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评估准分子激光创伤后野生型和基质溶解素敲除小鼠中细胞外基质组分的表达。 目标C的第二部分将仔细检查这些小鼠中角膜细胞外基质的MMP-7依赖性分解产物。 最后，使用MMP-7/ECM双敲除小鼠，将研究角膜透明度降低对特异性细胞外基质组分的需求。 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透明度机制的理解。 这些角膜研究的结果最终可能有助于理解其他器官的愈合。