Age-Related Differences in Cartilage OP-1

软骨 OP-1 与年龄相关的差异

• 批准号: 6894070
• 负责人: SUSAN CHUBINSKAYA
• 金额: $ 21.16万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-15 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6894070
• 关键词: SDS polyacrylamide gel electrophoresis; aging; antisense nucleic acid; articular cartilage; binding proteins; bone morphogenetic proteins; cartilage disorder; clinical research; enzyme linked immunosorbent assay; high performance liquid chromatography; human subject; human tissue; immunoprecipitation; in situ hybridization; interleukin 1; mechanical stress; neutralizing antibody; oligonucleotides; osteoarthritis; polymerase chain reaction; protein degradation; protein metabolism; protein structure function; tissue /cell culture; western blottings

Degeneration of articular cartilage occurs with aging and accelerated in osteoarthritis (OA). However, the mechanisms responsible for these changes are currently unknown. The focus of this project is the member of the bone morphogenetic protein (BMP) family, BMP-7 or osteogenic protein-1 (OP-1), that has an ability to induce anabolic processes in cartilage. Previously, we showed for the first time that OP-1 message and protein are expressed in adult human articular cartilage and that their levels declined with aging and cartilage degeneration. Our hypothesis is that, with aging, human articular cartilage exhibits a decrease in endogenous OP-1 content, synthesis and metabolism. This may lead to an elevated susceptibility of the chondrocytes to catabolic processes thus contributing/promoting cartilage degeneration. The long-term objective is to understand the biological function and regulation of endogenous OP-1 in aging human articular cartilage in comparison to OA. The uniqueness of this project is that we have unlimited access to human cartilages from organ donors collected through the Regional Organ Bank of Illinois. We can distinguish between normal (no-pathologically) and pathologically progressive (degenerative) aging by documenting changes in a joint that rarely develops OA (ankle joint) compared to changes in a joint with the higher prevalence of OA (knee joint). The objectives of the present study will be tested by three specific aims: 1) Assess the response of articular cartilage to catabolic stimuli (IL-1 and mechanical injury) by studying cartilage endogenous OP-1 and investigate whether this response is different between normal and pathologically progressive aging; 2) Investigate the metabolism of endogenous OP-1 at its near steady state and under exposure to catabolic stimuli (IL-1 and mechanical injury) in adult articular cartilage of different ages; 3) Study whether changes in endogenous OP-1 have a causative role in impairing cartilage homeostasis in normal and pathologically progressive aging. We intend to perturb OP-1 function or expressing using specific neutralizing antibody, binding proteins, and/or antisense oligonucleotides. In order to accomplish these specific aims, a novel sensitive ELISA method has been developed by us. This method allows us to analyze the concentrations of OP-1 protein in cartilage extracts and culture medium. Also a new neoepitope antibody to the specific cleavage site between pro- and mature domains of OP-1 has been developed and characterized. This antibody is critical, since both forms of OP-1 (pro- and mature) have been identified in human articular cartilage and the neoepitope antibody will provide a tool to assess changes in processing and activation of endogenous OP-1 under different experimental conditions. This study will contribute to the understanding of the mechanisms of cartilage repair/anabolism.

关节软骨的退化随着年龄的增长而发生，并在骨关节炎（OA）中加速。然而，导致这些变化的机制目前尚不清楚。该项目的重点是骨形态发生蛋白（BMP）家族的成员，BMP-7或成骨蛋白-1（OP-1），具有诱导软骨合成代谢过程的能力。在此之前，我们首次发现OP-1信息和蛋白在成人关节软骨中表达，并且其水平随着衰老和软骨退变而下降。我们的假设是，随着年龄的增长，人类关节软骨表现出内源性OP-1含量，合成和代谢的减少。这可能导致软骨细胞对分解代谢过程的敏感性升高，从而促成/促进软骨变性。长期目标是了解与OA相比，内源性OP-1在老化人关节软骨中的生物学功能和调节。这个项目的独特之处在于，我们可以无限制地从伊利诺伊州区域器官库收集的器官捐赠者那里获得人体软骨。我们可以通过记录很少发生OA的关节（踝关节）的变化与OA患病率较高的关节（膝关节）的变化来区分正常（非病理性）和病理性进行性（退行性）老化。本研究的目的将通过三个具体目标进行测试：1）评估关节软骨对分解代谢刺激的反应（IL-1和机械损伤），并研究这种反应在正常和病理性进行性衰老之间是否不同; 2）研究内源性OP-1在其接近稳定状态和暴露于分解代谢刺激下的代谢3）研究内源性OP-1的变化是否在正常和病理性进行性衰老中损害软骨稳态中具有致病作用。我们打算使用特异性中和抗体、结合蛋白和/或反义寡核苷酸干扰OP-1的功能或表达。为了实现这些特定的目标，我们开发了一种新的灵敏的ELISA方法。这种方法使我们能够分析软骨提取物和培养基中OP-1蛋白的浓度。还开发并表征了针对OP-1的原结构域和成熟结构域之间的特异性切割位点的新的新表位抗体。这种抗体是至关重要的，因为两种形式的OP-1（原和成熟）已被确定在人类关节软骨和新表位抗体将提供一种工具，以评估在不同的实验条件下内源性OP-1的加工和激活的变化。本研究将有助于理解软骨修复/再生长的机制。