The Study Of The Role Of Annexin A2 In DDR2-dependent Synovium Invasion In RA

Rheumatoid arthritis is a chronic, systemic to progressive, invasive, disabling arthropathy as the main manifestation of autoimmune disease. If it is not treated in time, the disease will be rapid development, causes the aggravation of articular cartilage injury, eventually leading to the loss of joint deformity, function, and has a high morbidity. Therefore, to clarify the related signal pathway involved in the joint adjacent bone, and cartilage injury in RA, we not only can find the key molecules and the regulation mechanism, bualso could put forward the new understanding for RA occurrence, and provide potential drug targets for the clinical treatment of RA.It has been reported that: the basic pathological feature of RA is involved in the whole body joint synovitis and pannus formation. The immune pathological components pannus in immune and erosion can be divided into two parts. The erosion part is mainly composed of osteoclasts adjacent bone, cartilage(Osteoclast) and synovial fibroblasts(Synovial Fibroblast, SF) form, synovial fibroblast cells including macrophage like synoviocytes and fibroblast like synoviocytes(Fibroblast-Like Synovial Cells, FLS). Matrix metalloproteinase FLS excessive secretion is thought to be arch-criminal RA bone, cartilage destruction. In the RA organization MMPs is directly involved in the degradation of articular bone, cartilage, type II collagen produced and can induce FLS secretion of MMPs, then formed the vicious spiral, aggravate the articular bone, cartilage damage.Our previous results find that: ① DDR2 is high expression in synovial tissue of patients with RA and FLS cells, and assumes the sustained activation state[3, 4]; ② the primary culture of RA FLS cells in patients with MMPs molecules that can [5] sustained high level secretion of MMP1, MMP13 and RA joint adjacent bone, cartilage pathology the direct damage related; ③ the DDR2 can be controlled by adjusting the AP1, Runx2 transcriptional control of MMP1, MMP13 and other MMPs molecules expression of [5]; ④ through the research of DDR2 receptor extracellular domain, we found that combined with the extracellular domain of DDR2 could be blocked by type II collagen and DDR2 can inhibit RA synovial cells secrete MMP-1[6]. In addition, we still in view of(collagen-induced arthritis, CIA) found on rat arthritis model, soluble DDR2 can improve the adjacent bone, cartilage RA joint injury.Despite we have done a lot with DDR2 in RA, in the course of the development of functional study as the breakthrough point, has carried on some research and made some achievements, but there are still many issues worthy of in-depth study: 1. DDR2 is distributed in the cell membrane of FLS surfaces with protein tyrosine kinase activity of the receptor, type II collagen makes DDR2 occurred after phosphate activation, it will be combined with which molecules? 2. This molecules is phosphorylated molecules or non phosphorylated molecules? 3. What are their functions? Can they regulate the expression of MMPs?To answers these questions help to clarify the DDR2 mediated RA FLS cell MMPS molecular mechanism of secretion, and system to uncover the role of collagen type II RA under the joint adjacent bone, cartilage injury mechanism. Therefore, based on the previous work, we plans from the following aspects: Firstly, research by DDR2 overexpression of lentivirus upregulation of FLS expression level of DDR2 in cells, with the interaction of DDR2 molecules as the breakthrough point, the molecular interaction using immunoprecipitation combined with SDS-PAGE method for screening, separation of DDR2, to determine the peptide mass spectrometry identity. The results combined with bioinformatics and related literature analysis, we obtained by screening DDR2 molecular interaction- vimentin(Vimentin) as the research object, co precipitation to determine the interaction between DDR2 and Vimentin using immune, confocal microscopy expression and distribution of Vimentin and DDR2 in FLS cells by laser. Second, then, we use RNAi, Real-time PCR, Western Blot and other experimental methods, the effects of DDR2 activation on Vimentin expression and phosphorylation state. And to study the function of Vimentin as the starting point, the effects of Vimentin on invasion, migration ability of MMPs and FLS cells. Thirdly, we expand the number of samples, with osteoarthritis(OA) in synovial tissue in patients with negative control, system on distribution, Vimentin expression in synovial tissue of patients with RA, and further to evaluate its clinical significance.Through the experiment, we get the following results: ① By immunoprecipitation method combined with SDS-PAGE separation, protein interaction obtained 8 DDR2, after analysis and peptide mass spectrometry and bioinformatics, identified 8 protein identity, we choose Vimentin as the object of study the next step; ② In HEK293 T cells, the co immunoprecipitation method, confirmed the interaction between DDR2 and Vimentin activation; ③ Confocal microscopy revealed that DDR2 and Vimentin were colocalized relationship in RA FLS cells by using laser;④ RA FLS of DDR2 on the cell membrane after activation by collagen type II, change of the expression level of Vimentin had a little, but Vimentin phosphorylation levels significantly increased;⑤ The phosphorylation of Vimentin can promote the expression of MMP13 and RA activation in FLS; ⑥ Transwell assay and cell scratch assay display that the phosphorylation of Vimentin activated RA FLS can promote cell invasion, migration;⑦ In expanding the sample size, in synovial tissue of patients with OA as control, the expression level of phosphorylation of Vimentin and water in the synovial tissue of RA patients was higher than that of the average OA synovial tissue.