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Targeted Cleavage of Matrix Molecules in a Neocartilage

新软骨中基质分子的靶向裂解

基本信息

批准号：
8141942
负责人：
JACK L LEWIS
金额：
$ 18.77万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-10 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8141942
关键词：
Animals Biochemical Candidate Disease Gene Cartilage Cartilage Matrix Chondrocytes Cleaved cell Collagen Collagen Fibril Connective Tissue Consensus Degenerative polyarthritis Dermatan Sulfate Development Digestion Disease Elements Enzymes Evaluation Extracellular Matrix Extracellular Matrix Proteins Failure Fracture Genes Genetic Genome Glues Goals Growth Indium Knockout Mice Laboratories Location LytA enzyme Measures Mechanics Methods Minor Modeling Motivation Peptide Hydrolases Pharmaceutical Preparations Prevention Process Property Proteins Proteoglycan Research Site Specificity Staging System Techniques Therapeutic Intervention Thrombin Tissue Engineering Tissues Translating Validation Work aggrecan cartilage cell chondroitinase B decorin disability drug development experience fibromodulin interest mouse model overexpression prevent public health relevance repaired single molecule success tool treatment strategy

项目摘要

DESCRIPTION (provided by applicant): The goal of this project is to develop a method and model in which a chosen extracellular matrix protein of a tissue grown from cartilage cells (neocartilage) can be cleaved at a specific site. Addition of a translated thrombin protease recognition site will be inserted into the genetic sequence of an extracellular matrix molecule, the genetically modified molecule expressed by chondrocytes, and a neocartilage generated. The genetically modified protein could be cleaved by the addition of thrombin at any point in the development of the tissue. The immediate application is to allow evaluation of candidate structural molecules, molecules that carry load, by comparison of the mechanical properties of the neocartilage before and after cleaving the molecule. If the candidate molecule is a structural molecule, the mechanical properties should be reduced after cleavage of that molecule. The motivation for developing the method is to understand the mechanical failure process in osteoarthritis, where extracellular matrix molecules are digested by lytic enzymes and subsequently the tissue fails under relatively normal loads. Current strategies for disrupting the disease process involve search for agents to block critical molecules in the process. A significant challenge in this approach has been identifying the critical molecules to block. The traditional approach to identifying structural molecules is by digesting candidate molecules with an exogenous enzyme and measuring mechanical properties before and after. The problem with this approach is that there are very few enzymes available that are single molecule specific. The proposed method would allow digestion (cleavage) of a chosen molecule with specificity, greatly enlarging the number of matrix molecules that could be evaluated as candidate structural molecules. The method may have other applications in tissue engineering and drug release. The approach would be a valuable tool in understanding the OA disease process and as a guide for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is the largest cause of disability, but there is poor understanding of the mechanisms of the disease process, particularly how cartilage is degraded. This project will develop methods to identify how load is transmitted through cartilage, and which specific load carrying molecules might be damaged during the OA process. Identification of these molecules would allow development of drugs that could retard the damage and provide a disease modifying treatment to OA.

描述（由申请人提供）：本项目的目标是开发一种方法和模型，其中可以在特定位点切割从软骨细胞（新软骨）生长的组织的选定细胞外基质蛋白。添加翻译的凝血酶蛋白酶识别位点将被插入细胞外基质分子的基因序列中，该基因修饰的分子由软骨细胞表达，并产生新软骨。在组织发育过程中的任何时候，通过加入凝血酶，基因修饰的蛋白质都可以被切割。直接的应用是通过比较切割分子之前和之后的新软骨的机械特性来评估候选结构分子，即承载载荷的分子。如果候选分子是结构分子，则在该分子裂解后，机械性能应降低。开发该方法的动机是了解骨关节炎中的机械失效过程，其中细胞外基质分子被溶解酶消化，随后组织在相对正常的负荷下失效。目前破坏疾病过程的策略包括寻找阻断过程中关键分子的药物。这种方法的一个重大挑战是确定要阻断的关键分子。识别结构分子的传统方法是通过用外源酶消化候选分子并测量前后的机械性能。这种方法的问题是，只有很少的酶是单分子特异性的。所提出的方法将允许特异性地消化（切割）所选择的分子，大大增加可以作为候选结构分子进行评估的基质分子的数量。该方法可能在组织工程和药物释放中具有其他应用。该方法将是一个有价值的工具，在了解OA疾病的过程中，并作为指导治疗干预。公共卫生相关性：骨关节炎（OA）是残疾的最大原因，但对疾病过程的机制，特别是软骨如何降解的了解甚少。该项目将开发方法来确定载荷如何通过软骨传递，以及在OA过程中哪些特定的载荷携带分子可能会受损。这些分子的鉴定将允许开发可以延缓损伤并为OA提供疾病修饰治疗的药物。