Seprase调控体系在类风湿关节炎发生发展中的作用及解毒通络法对其的干预机制

Seprase is believed to be directly related with Rheumatoid Arthritis. Currently, the researches on it are not only very less but also fully debatable. However, the latest report published in Science and other magazines shows the Seprase involvement in immune escape and hence provides an entirely new clue. This study intends to build gene knocked-out mouse model research platform to explore the role of Seprase regulating system in RA and other possible mechanism; side by side, under the guidance of Chinese Medicine Arthralgia theory along with reconstructed model of collagen-induced lipopolysaccharide enhanced arthritic animal in cellular microenvironment, tries to simulate the detoxification & dredging therapy for wind-damp-hot arthralgia using Qingbi Decoction as a carrier and probe out the intervention of detoxification & dredging therapy in the process of articular changes in RA with Seprase related mechanism. We designed an in vitro and in vivo experiment, using techniques such as reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, in situ hybridization, immunofluorescence, immunoblotting, ELISA, flow cytometry etc to verify the expression levels and linkages of dipeptidyl-aminodipeptidaseIV (DPP IV)/CD26, myeloid-related protein (MRP), protease-activated receptor (PAR), monocyte chemoattractant protein (MCP), follistatin-related protein (FRP), downstream genes , enzymes & inflammatory factors etc in the Seprase regulating system. The study design is based on the immune escape mechanism to clarify the anti-RA mechanism of Chinese Medicine as well as search a new RA therapeutic target.

Seprase被认为与类风湿关节炎（RA）有直接联系，但目前在RA中调控机制研究较少且存在争议；而Science等杂志最新报道的Seprase参与免疫逃逸，则提供了全新线索。本研究拟构建Seprase基因敲除小鼠研究平台，探索Seprase调控体系在RA中的作用及可能机制；并在中医痹证理论的指导下，复制胶原诱导并脂多糖加强的类风湿关节炎动物模型、细胞微环境，模拟解毒通络法辨证治疗的风湿热痹，以清痹方为研究载体，探讨解毒通络法干预RA关节病变进程的Seprase相关机制。采用体内、外实验，RT-PCR、免疫组化、原位杂交、免疫荧光、免疫印记、ELISA、流式细胞术等技术方法，拟探明Seprase调控体系中二肽基肽酶、髓系相关蛋白、蛋白酶活化受体、单核细胞趋化蛋白、卵泡抑素相关蛋白等上、下游基因和相关酶、炎性因子的表达及其联系。旨在基于免疫逃逸机制阐明中药对抗RA机理，寻找全新的RA治疗靶点。

Seprase 被认为与类风湿关节炎（RA）有直接联系，但目前在RA 中调控机制研究较少且存在争议；而Science 等杂志报道的Seprase 参与免疫逃逸，则提供了全新线索。本课题以“毒痹论”为指导思想，构建Seprase 基因敲除小鼠研究平台，探索Seprase 调控体系在RA 中的作用及可能机制；并在中医痹证理论的指导下，复制胶原诱导并脂多糖加强的类风湿关节炎动物模型、细胞微环境，模拟解毒通络法辨证治疗的风湿热痹，以清痹方为研究载体，探讨解毒通络法干预RA 关节病变进程的相关机制。实验结果显示，在CIA的发病过程中，Seprase、TGFβ1、IL–10、突变型P53等免疫逃逸机制的激活，同时影响其上下游相关炎性因子、相关酶，以及细胞增殖、迁移相关因子或生发中心等区域结构。以Seprase、TGFβ1为核心机制的复杂网络机制失调，直接导致类风湿关节炎的发生、发展。以解毒通络为主法的清痹方对CIA模型动物关节症状、影像学改变具有良好的改善作用，能有效抑制相关基因、蛋白的表达，提高有利因子的表达水平，从而调节Seprase–TGF网络中失衡的免疫机制。中药各剂量组间有一定的量效关系。高剂量解毒通络中药对CIA模型的治疗作用与MTX接近。由此可见，解毒通络法治疗类风湿关节炎作用明确，其作用机理与Seprase调控网络相关，同时也显示了Seprase可能作为RA治疗靶点的新药开发潜力。