NKp46+NK细胞调控胶原诱导关节炎DBA/1小鼠关节滑膜炎和骨破坏的免疫机制及小乌桂颗粒剂对其干预的研究

Rheumatoid arthritis (RA) is a common autoimmune disease with the sustained progression of symmetrical synovitis in peripheral joints and generally leads to the destruction of articular cartilage and bone. Previous studies have revealed that nature killer (NK) cells have involved in the development of RA. Applicant’s previous studies found the NKp44+NK cells were high expressed in peripheral blood and joints in patients with RA, which promoted the proliferation of synovium by secreting IL-22. However, the exact immune mechanism of NKp44+NK cells in RA pathogenesis is yet to be studied. Although the Traditional Chinese medicine, Xiaowugui granule, has been found that it might ease symptoms caused by RA both in clinical and experimental research, the related immune mechanism remains to be clarified. Therefore, based on the preliminary research, the following experiments were designed. First, establish the collagen-induced arthritis (CIA) model in DBA/1 mouse and dynamically evaluate the expression and function of NKp46+NK cells in the model. Second, in order to clarify the role of NKp46+NK cells in the development of CIA, establish the CIA model with over expression and defective NKp46+NK cell by adoptive transfer and antibody elimination technology. Third, separate the fibroblast-like synoviosytes and CD14+ monocytes, and then co-culture those cells with NKp46+NK cell respectively to elucidate the immune mechanism of NKp46+NK cells in joint inflammation and bone destruction. Fourth, use Xiaowugui granule as drug intervention to CIA model with over expression of NKp46+NK cells, and clarify its possible mechanism in relieving RA. All of those studies may provide the foundation for the clarification of immune regulation mechanism in RA and the development of new therapeutic drug.

类风湿关节炎（RA）是以外周对称性关节滑膜炎并导致骨和软骨破坏为特征的自身免疫病，NK细胞及亚群参与了疾病的进展。申请者前期发现RA外周血及关节中NKp44+NK细胞高表达，并通过表达IL-22促进RA滑膜增殖，但该细胞群在滑膜炎症和骨破坏中作用地位和机制尚不清楚。小乌桂颗粒的临床和基础研究发现其能够缓解RA关节炎，但相关免疫机制有待阐明。为此本项目拟：①建立DBA/1小鼠CIA模型，动态观察NKp46+NK细胞表达及功能；②过继回输/抗体剔除构建NKp46+NK细胞过表达/缺陷CIA模型，明确该细胞在CIA关节炎发生中的作用；③建立NKp46+NK细胞分别与FLS及CD14+单核细胞的共培体，阐明NKp46+NK细胞在关节炎症及骨破坏中的分子机制；④小乌桂颗粒干预NKp46+NK细胞过表达CIA模型，明确其缓解关节炎的免疫机制。从而为RA发病机制的进一步阐明及药物研发提供新的证据和靶标。

类风湿关节炎（RA）以滑膜炎及骨破坏为特征，多种免疫细胞可能参与RA的发生发展，自然杀伤（NK）细胞及其亚群NKp46+NK细胞作为重要的一群免疫细胞，其在RA发病中的作用有待进一步阐明。为此本项目：①建立小鼠胶原诱导关节炎（CIA）模型，应用流式细胞术和免疫荧光动态观察CIA小鼠不同组织中NK细胞及其亚群的比例，并分析其与关节炎严重程度的相关性。②过继回输构建NKp46+NK细胞过表达的小鼠CIA模型，评估小鼠关节炎发病率和严重程度，免疫荧光检测NKp46+NK细胞在不同组滑膜组织中的表达。③应用高中低剂量小乌桂颗粒剂及对照药干预NKp46+NK细胞过表达的小鼠CIA模型，评估小鼠关节炎严重程度、流式细胞术和免疫荧光检测小鼠外周血/足爪以及关节滑膜组织NKp46+NK细胞表达。研究结果显示：NKp46+NK细胞在CIA小鼠不同组织中均有表达，特别在炎症关节组织中高表达，且升高的NKp46+NK细胞与小鼠关节炎指数、关节炎病理及关节骨破坏评分呈正相关；项目在CIA小鼠模型的基础上成功构建了NKp46+NK细胞过表达的小鼠CIA模型，过继回输的NKp46+NK细胞在CIA小鼠炎症关节中聚集，并促进了CIA小鼠的发病，加重了小鼠滑膜炎和骨破坏；小乌桂颗粒剂干预研究发现其能够下调外周血和关节组织中的NKp46+NK细胞比例，具有缓解CIA小鼠关节炎症的疗效。本研究阐明了NKp46+NK细胞在CIA小鼠关节滑膜炎症和骨破坏进展中的重要作用，明确了小乌桂颗粒剂通过下调NKp46+NK细胞缓解CIA小鼠关节炎症的免疫学机制。过继回输NKp46+NK细胞的小鼠模式，为DBA/1小鼠CIA加强模型的制备提供新的技术手段，可能为RA免疫机制研究提供新的改进型动物模型；研究发现的小乌桂颗粒剂靶向NKp46+NK细胞防治RA的机制，为小乌桂颗粒剂的临床应用进一步提供了理论依据。研究成果在投SCI论文2篇，其中1篇已经由Frontiers in Immunology杂志（IF=7.56）接收；协助培养在读博士研究生1名，独立培养在读硕士研究生2名；在本项目研究基础上，NK细胞亚群（ILCs）在RA发病中的进一步免疫机制研究获得国家自然科学基金面上项目延续资助。