类风湿关节炎NKp44细胞协同MDSCs诱导OCs分化及中药“骨灵丸”对其干预机制研究

Rheumatoid arthritis (RA) is a common autoimmune disease and often leads to the bone destruction in the joint. The applicant previously presided studies subsidized by the Natural Science Foundation of China and Guangdong Province had concluded that the regulation of RANKL-RANK-OPG on osteoclast (OCs) was the main mechanism during the bone destruction in patients with RA. Traditional Chinese medicine guling pellet has shown their therapeutic effect of bone protect in RA. But the regulatory mechanisms of guling pellet to RANKL-RANK-OPG system and immune cells (including MDSCs and NKp44 cells or NKp46+NK cells) are yet to be studied. Therefore, based on the preliminary research foundation, we design the experiment below. First, separate the MDSCs and NKp44 cells from RA patients, and then establish the co-culture system of those cells in order to explicit the interaction mechanism in process of OCs differentiation. Second, establish the animal model of RA, collagen-induced arthritis (CIA) model, and then deplete these two cells in order to confirm the role of those cells in bone destruction in the CIA model. Third, intervene the CIA model with guling pellet and clarify its bone protect mechanism during the therapy of RA. The aim of this study is to clarify the mechanism of MDSCs and NKp44 cells in RA bone destruction and to confirm the bone protect mechanism of guling pellet. All of those studies may provide the foundation for the clarification of immune regulation mechanism in RA bone destruction and the development of new therapeutic drug.

类风湿关节炎（RA）是一种常见自身免疫疾病，常导致关节骨破坏。申请者前期承担的国家和广东省自然基金研究表明：RANKL-RANK-OPG系统调控破骨细胞（OCs）活化是RA骨破坏的主要环节，中药骨灵丸对RA有良好护骨疗效，但相关免疫细胞（MDSCs和NKp44/NKp46+NK细胞）对该系统的调节机制及骨灵丸的护骨内涵尚有待深入研究。在以上工作基础上，本项目拟：①建立RA MDSCs和NKp44细胞共培体，分析两者作用下OCs分化规律；②建立剔除MDSCs和/或NKp46+NK细胞的RA动物模型CIA，研究两者通过OPG系统调控OCs促CIA骨破坏的机制；③运用骨灵丸干预CIA，进一步丰富其通过NKp44细胞协同MDSCs诱导OCs分化的护骨作用内涵。总体目标是阐明MDSCs/NKp44细胞调控OCs骨破坏机制及丰富骨灵丸护骨作用内涵，为揭示RA骨破坏免疫调控机制和新治疗药物研发奠定基础。

探索类风湿关节炎（RA）骨破坏机制并阻断其进程，是目前RA基础研究和临床治疗的重要目标。已知RANKL-RANK-OPG系统调控破骨细胞（OCs）活化是RA骨破坏的主要环节，中药骨灵丸对RA有良好护骨疗效，但相关免疫细胞，包括髓源性抑制细胞（MDSC）和NKp46+NK细胞对该系统的调节机制及骨灵丸的护骨内涵尚有待深入研究。本项目通过纳入RA患者、应用基因工程小鼠（SKG小鼠，NKp46基因敲除小鼠），通过体外细胞学实验及体内小鼠实验研究了MDSC及NKp46+NK细胞在类风湿关节炎发病机制中的作用以及骨灵丸的护骨机制。研究取得了以下创新性结果：①风湿病患者MDSC的扩增与关节炎相关；②M-MDSC通过分化为破骨细胞加剧RA骨破坏，选择性剔除M-MDSC能够缓解胶原诱导性关节炎小鼠关节炎症及骨破坏；③NKp46受体能够抑制MDSC，尤其是G-MDSC的扩增；④骨灵丸的骨保护机制可能与降低CIA小鼠M-MDSC的扩增、改善RANKL/OPG比值、降低炎性细胞因子TNF-α、IL-1β、IL-6的表达有关。本研究发现了RA治疗的新的靶细胞：M-MDSC；围绕RANKL-RANK-OPG系统，进一步揭示了骨灵丸骨保护的作用机制，为其临床应用推广奠定了基础。