镁调节软骨细胞OPN表达和功能影响软骨钙化在骨关节炎中的作用及其机制研究

Osteoarthritis (OA) is one of the most common diseases of aging people, the pathogenesis of which is still not very clear. Applicant’s study found that magnesium (Mg), osteopontin (OPN) and cartilage calcification are closely related to occurrence, development and pain symptoms of OA; Mg is related to cartilage calcification; Mg can promote the OPN expression in chondrocyte. Combined with literature reports, the up-regulated expression of OPN induced by Mg could inhibit calcification of vascular smooth muscle cells; Mg can modulate the function of matrix protein by promoting its binding with integrin. It could be speculated that Mg can promote the expression of OPN (a kind of matrix protein) in chondrocyte to inhibit cartilage calcification, thereby protect articular cartilage and alleviate pain symptoms. This project intends to elucidate the role of Mg and cartilage calcification in occurrence and development of OA and their influence on pain symptoms, to confirm that the inhibitory role of Mg in cartilage calcification is based on OPN, through both experiments of cells in vitro and animals in vivo using siRNA interference, gene overexpression and CRISPR/Cas9 gene editing technologies, and then perform a prospective cohort study to elucidate whether knee joint cartilage calcification can promote the occurrence, development and pain symptoms of knee OA in the general population, finally further confirm the prevention and treatment effect of Mg from the perspective of cartilage calcification and provide a new idea for the clinical prevention and treatment of OA.

骨关节炎（OA）是一种常见关节退行性疾病，发病机理仍不十分明确。申请人研究发现：镁、骨桥蛋白（OPN）和软骨钙化与OA发生发展及疼痛症状密切相关，而镁与软骨钙化相关并可上调软骨细胞OPN表达。结合文献报道：镁可上调OPN表达从而抑制血管平滑肌细胞钙化，镁还可通过促进基质蛋白与整合素结合而调节其功能，提出假说：镁可调节软骨细胞OPN（一种基质蛋白）表达和功能，从而抑制软骨钙化，最终保护关节软骨并缓解疼痛症状。本项目拟采用siRNA干扰、基因过表达和CRISPR/Cas9基因编辑等技术，在体外细胞（单层和三维培养）与活体动物两个层面，明确镁和软骨钙化对OA发生发展及疼痛症状的影响，证实镁通过调节OPN表达和功能影响软骨钙化，并通过前瞻性队列研究进一步明确膝关节软骨钙化可促进人群膝OA发生发展并产生疼痛。从抑制软骨钙化的视角明确镁对OA的预防和治疗作用，为OA临床防治提供新思路。

关节炎（OA）是一种常见关节退行性疾病，发病机理仍不十分明确。课题组研究发现：镁、骨桥蛋白（OPN）和软骨钙化与OA发生发展及疼痛症状密切相关，提出假说：镁可调节软骨细胞OPN表达从而抑制软骨钙化，最终保护关节软骨并缓解疼痛症状。为验证这一假说，课题组通过动物实验证明了低镁干预可减少OA小鼠软骨OPN的表达，促进软骨钙化并加重OA小鼠的疼痛症状；OPN敲除小鼠OA建模后钙化相关指标表达增多、炎症加重且疼痛症状更加明显。在动物实验的基础上，课题组通过体外细胞实验证明了低镁可减少小鼠软骨OPN的表达，促进软骨细胞钙化；OPN敲除小鼠软骨细胞转录组测序发现OPN敲除后主要发生变化的生物学效应包括矿化、成骨细胞分化、免疫系统反应、疼痛行为反应等。以上研究结果提示低镁可通过上调OPN表达促进软骨钙化。课题组进一步深入研究了钙化对OA的影响，通过体外细胞实验，将成功诱导钙化的软骨细胞以及未诱导钙化的软骨细胞分别与正常软骨细胞共培养，发现钙化的软骨细胞可减少软骨细胞外基质合成，促进软骨细胞外基质降解及炎症反应；将成功诱导钙化的软骨细胞以及正常软骨细胞分别与白细胞介素1（IL-1）干预与否正常软骨细胞共培养，发现钙化的软骨细胞受到炎症刺激后OA表型更加明显，提示软骨钙化可能是OA的危险因素。此外，课题组通过人群研究证实了低血镁人群患有膝关节软骨钙化的比例更高，且与血镁浓度呈剂量依赖性。以上研究结果提示Mg可调节软骨细胞OPN表达从而抑制软骨钙化，最终保护关节软骨、延缓OA发生发展并缓解疼痛症状。本课题从抑制软骨钙化的视角明确镁对OA缓解作用，为OA临床防治提供新思路。