生长分化因子5抑制骨关节炎软骨破坏的机制研究

Osteoarthritis (OA), characterized by degeneration of articular cartilage, is the most common form of human arthritis and a major concern for aging societies worldwide. Growth and differentiation factor 5 (GDF5) is a bone morphogenetic protein involved in cartilage development and joint formation. A promoter polymorphism (rs143383) in GDF5 has been found to be strongly associated with both hip and knee OA, and is the most widely replicated genetic association with knee OA. This variant is functional, with the lower gene expression variant having increased genetic risk. However, the underlying pathogenesis is largely unknown. Our previous study showed GDF5 effectively suppressed catabolic factors such as MMP, ADAMTS and NOS2 in IL-1β-treated chondrocytes and microRNA-17 is likely to be involved in this process..In this proposal, we will perform experiments to investigate the modulation effect of GDF5 on cartilage homeostasis and the underlying mechanism. Firstly, we attempt to analyze the association of GDF5 expression in articular cartilage of human and animal models with OA occurrence. Secondly, we will investigate the essential roles of GDF5 in maintaining cartilage homeostasis and inhibiting osteoarthritic cartilage destruction in OA animal models. Finally, we will use technical methods such as miRNA interference, gene and protein assay, microarray and 3’UTR reporter gene to illustrate miRNA-17, which was induced by GDF5, as a direct inhibitor of these catabolic enzymes. Thus, the project may provide a scientific basis for developing GDF5 and miR-17 as novel therapeutic targets for treatment of OA.

骨关节炎（Osteoarthritis，OA）是一种常见的慢性关节疾病，其主要病变是关节软骨的退行性变。生长分化因子5（GDF5）参与调控软骨发育和关节形成，多项遗传学研究显示GDF5启动子区域的SNP（rs143383）是髋、膝关节OA的危险因素，然而其中的致病机制还有待深入研究。我们前期研究发现，GDF5作用于软骨细胞，能有效抑制被IL-1β激活的MMP、ADAMTS、NOS2等多种分解因子，并且microRNA-17极有可能参与了此调控过程。鉴于此，本项目拟分析人类和动物模型的关节软骨中GDF5的表达与OA的相关性；通过OA动物模型证明GDF5在维持关节软骨稳态及抑制OA软骨破坏中的关键作用；应用miRNA干扰、mRNA和蛋白检测、基因芯片、3’UTR报告基因等技术手段阐明GDF5调控miR-17抑制软骨分解代谢的机制。项目成果对于OA发病机制的研究及探求新的治疗手段具有积极的意义。

项目背景：骨关节炎是一种常见的慢性关节疾病，其主要病变是关节软骨的退行性变。生长分化因子-5（GDF-5）参与调控软骨发育和关节形成，多项遗传学研究显示GDF5启动子区域的SNP（rs143383）是髋、膝关节骨关节炎的危险因素。本实验观察GDF-5对小鼠骨关节炎的治疗效果以及探讨GDF-5对软骨细胞表型的调控作用及其调控机制。.研究内容：体内实验通过手术切断内侧半月板胫骨韧带，构建小鼠骨关节炎模型并在术后4周行关节腔内注射GDF-5，每周1次，共4次。体外试验将原代培养的小鼠关节软骨细胞分为5组，对照组包括未加处理的空白组、IL-1β (5 ng/ml)组，实验组包括3个浓度梯度 (20、100、300 ng/ml) GDF-5分别与IL-1β (5 ng/ml)共作用组，作用时间为24 h。用逆转录-定量聚合酶链反应检测合成基因及基质金属蛋白酶(MMP)-3、MMP-12、MMP-13、一氧化氮合酶-2(NOS2)和含Ⅰ型血小板结合蛋白基序的解聚蛋白样金属蛋白酶(ADAMTS)-5等分解基因表达变化。然后筛选GDF-5反应性miRNA，通过抑制及过表达miRNA来研究其在GDF-5调控中的作用，并使用miRNA关节腔内注射治疗小鼠骨关节炎。最后检测GDF-5及相关miRNA在人骨关节炎软骨中的表达。.结果：GDF-5在小鼠骨关节炎软骨中表达减低。关节腔内注射GDF-5能减轻骨关节炎的严重程度，阻止关节软骨破坏。应用IL-1β刺激体外培养的关节软骨细胞，MMPs、ADAMTS5、NOS2等高表达，同时合成基因表达降低。当IL-1β作用体系中加入一定量的GDF-5后，分解基因表达均得到显著抑制，合成基因表达逆转。机制研究发现GDF5通过调控miR-17能抑制软骨分解因子的表达，MMP-3、MMP-13、NOS2和ADAMTS-5均是miR-17作用的靶基因。关节腔内注射miR-17能延缓骨关节炎的进展。人类骨关节炎软骨中GDF-5表达与miR-17表达具有相关性，它们表达下调与骨关节炎软骨破坏有相关性。.科学意义：本研究首次揭示了GDF-5对miR-17具有调控作用，miR-17的上调对于缓解或治疗骨关节炎具有显著的效果。因此，miR-17可以作为骨关节炎的新的治疗靶点。