Annexin 5过度表达致关节软骨细胞终末分化机制研究

Osteoarthritis (OA) is the most common type of degenerative joint diseases. It is a common chronic, progressive musculoskeletal disorder characterized by gradual loss of articular cartilage. OA is regarded as a complex disease whose cause is not completely understood yet, and so far there is no cure for OA. Recent studies revealed that Annexin 5 was highly expressed in the OA chondrocytes, while not in the healthy ones. And then the terminal differentiated markers, such as Apase, MMP13 and Runx-2 of the normal chondrocytes were also upregulated by the Annexin 5 vector transfected chondrocytes from our preliminary data. All those data was indicating the chondrocytes in the status of terminal differentiation, which loss the biological function of the chondrocyte. However, until now there was no research focus on the involved signaling mechanism of Annexin 5. Our preliminary data demonstrated the Wnt luciferase activity was inhibited, and simultaneously NF-kB signaling was activated in the overexpressed Annexin 5 groups. Depletion of Annexin 5 could be a considered and possible method to preserve the chondrocytes biological function out of the terminal differentiation, or the challenge from the OA. This program to investigate the mechanism of Annexin 5 leading articular chondrocytes to terminal differentiation. In this study, use the Annexin 5 gene knock-out mouse model, and observe the signaling pathway changes in vitro and vivo. Effecting on the Wnt and NF-kB pathways of the Annexin 5 may reveal its great role in the OA progress. Inhibiting Annexin 5 could be the next therapeutic target for OA treatments in future.

骨性关节炎(Osteoarthritis，OA)是一种发生机制不清，尚无有效防治手段的高发疾病。前期研究发现Annexin 5（A5）是一种可控细胞内、外钙离子浓度的胞浆蛋白，正常软骨细胞中无A5存在，而在OA患者软骨细胞中A5则大量聚集，但两者是否有相互作用尚无研究涉入；本课题组预实验提示过度表达A5的正常软骨细胞其终末分化相关基因表达明显上调，且胞内Wnt信号被过度抑制，而NF-kB通路明显激活；结合A5最新研究进展，提出A5对双钙离子通道的调控，导致Wnt通路抑制和NF-kB通路激活，进而导致了软骨细胞的终末分化，最终形成OA，且OA特殊内环境状态又维持了A5过度表达的假说；本项目拟用正常及A5基因敲除小鼠进行体内OA模型诱导及体外细胞与组织培养的方法，探索并证实A5在OA发生、发展过程中的重要作用，对阐明A5作用以及揭示OA机制有重要意义，为OA的防治提供一种新思路和理论基础。

本项目对在体和离体小鼠软骨细胞中，研究及探讨Annexin 5致软骨细胞终末分化的分子机制。首先实验以正常软骨细胞为研究对象，构建Annexin 5全长质粒及Annexin 5的siRNA，通过基因转染方法使得细胞内出现Annexin 5过表达或受抑制状态，然后通过提取RNA和细胞裂解液方式分别观察细胞内Annexin 5过表达及抑制时细胞相关基因变化，并分别在体外培养过程中加载IL-1β模拟OA内环境刺激，记录相关数据进行比对，发现体外刺激后Annexin 5基因过表达；其次，构建Annexin 5基因敲除小鼠，继而在该平台上分别进行体外活体组织培养，发现正常组较敲除组细胞外基质流失增多；手术建立小鼠体内OA模型，通过Western Blot及免疫染色等实验方法，观察基因敲除后Wnt/β–catenin和NF-kB等通路信号改变，发现Annexin 5过度表达激活NF-kB通路，同时抑制Wnt/β-catenin通路，导致关节软骨细胞终末分化，而IL-1β等终末产物又维持了Annexin 5的过度表达状态，从而使一种正反馈继续进行；体内OA模型发现，正常组OA软骨破坏程度不断加剧，而Annexin 5基因敲除组反而对软骨细胞起到保护作用；综上所述，抑制Annexin 5或为未来OA防治的提供新策略及理论基础。