S100A4通过mTOR促进类风湿关节炎成纤维样滑膜细胞介导血管生成的机制研究

Background: Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease characterised by hyperplasia of fibroblast-like synoviocytes and variable degrees of bone and cartilage erosion, leading to impairment of joint function. The pathogenesis remains to be fully elucidated. Angiogenesis is a prominent feature of RA.With regards to angiogenesis and.vasculogenesis, vascular endothelial growth factor (VEGF) is of outstanding importance.VEGF is probably the key regulator of neovascularization in inflammation. Serum VEGF concentrations are elevated in RA and correlate with disease activity. Furthermore, serum VEGF measured at first presentation in RA is highly significantly correlated with radiographic progression of disease over the subsequent year. Synovial angiogenesis results from increased level of VEGF plays a crucial role in the pathogenesis of RA. Rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) are active drivers of joint destruction in RA. It is important to identify factors responsible for the increased synthesis and secretion of VEGF by RAFLSs. Recent studies found that S100A4 is expressed at site of invasion in RA synovium and associated with the disease activity of RA, and mammalian target of rapamycin (mTOR) signaling is activated in synovial membrane of patients with RA. .Pilot study: Our pilot studies found that S100A4 increased the expression of VEGF and the phosphorylation of pS6, the substrate of mTOR in RAFLSs in a time-dependent and dose-dependent manner. .Hypothesis: The increased level of S100A4 in RA patients might lead to the activation of mTOR, which results in the increased secretion of VEGF from RAFLSs. The increased VEGF promotes synovial angiogenesis, which finally contributes to RA development..Objective: To determine the effect of S100A4-induced VEGF expression by RAFLSs and the mTOR signaling pathway involving in this process..Methods: Through clinical specimen, in vitro cell culture and RA mice model, we will investigate the mTOR signaling pathway involving in the S100A4-induced VEGF expression by RAFLSs..Conclusion: Through molecular, cellular, and animal models, our discovery hopes to determine the novel pathogenesis of RA and find possible new drug target for therapy.

类风湿关节炎（RA）是一种自身免疫性疾病。血管翳是引起RA关节病变、软骨破坏的主要原因及病理基础，血管生成是形成和维持血管翳的重要因素， VEGF是血管生成的关键调控因素。既往研究发现S100A4在RA患者体内蓄积，哺乳动物雷帕霉素靶蛋白（mTOR）信号通路在RA患者滑膜组织激活。我们预实验提示在原代培养的类风湿关节炎成纤维样滑膜细胞（RAFLSs），S100A4可激活mTORC1信号通路且呈浓度和时间依赖效应，同时S100A4可刺激RAFLSs分泌VEGF， 提示S100A4可能是通过激活mTOR信号通路促进RAFLSs分泌VEGF，进而调控血管生成。本项目拟通过临床标本、原代分离培养的RAFLSs及RA小鼠模型，从分子、细胞及动物整体水平深入探讨mTOR信号通路在S100A4促进RAFLSs介导血管生成的作用及其分子机制，以期为RA的发病机制提供新理论，为临床治疗RA提供新靶点。

类风湿关节炎（RA）是一种自身免疫性疾病，血管翳是引起RA关节病变、软骨破坏的主要原因及病理基础，血管生成是形成和维持血管翳的重要因素， VEGF是血管生成的关键调控因素。既往研究发现S100A4在RA患者体内蓄积，哺乳动物雷帕霉素靶蛋白（mTOR）信号通路在RA患者滑膜组织激活。我们预实验提示在原代培养的类风湿关节炎成纤维样滑膜细胞（RAFLSs），S100A4可激活mTORC1信号通路且呈浓度和时间依赖效应，同时S100A4可刺激RAFLSs分泌VEGF，提示S100A4可能是通过激活mTORC1信号通路促进RAFLSs分泌VEGF，进而调控血管生成。本项目拟通过临床标本、原代分离培养的RAFLSs及RA小鼠模型，从分子、细胞及动物整体水平深入探讨mTORC1信号通路在S100A4促进RAFLSs介导血管生成的作用及其分子机制。在病例对照研究中，免疫组化法证实S100A4及VEGF在RA患者滑膜组织中呈高水平表达，二者之间呈正相关。体外S100A4干预RAFLSs细胞模型发现，rhS100A4促进RAFLSs分泌VEGF、促进VEGF蛋白水平表达及激活mTORC1（S6、p-S6；S6K1、p-S6K1；4EBP-1、p-4EBP-1；AKT、p-AKT）信号通路均呈时间和剂量依赖正相关，rhS100A4孵育RAFLSs所形成的条件培养液诱导HUVECs体外形成管腔，mTORC1信号通路阻断剂雷帕霉素显著抑制rhS100A4促进RALFSs VEGF分泌、VEGF蛋白水平表达及mTORC1（S6、p-S6；S6K1、p-S6K1；4EBP-1、p-4EBP-1；AKT、p-AKT）信号通路激活，提示S100A4通过mTORC1信号通路刺激RAFLSs分泌VEGF、促进VEGF蛋白水平表达。项目从临床标本及体外细胞干预模型证实在RA病程中S100A4经mTORC1信号通路促进RAFLSs VEGF活性提高、VEGF蛋白水平表达，参与血管翳形成。通过对大数据TCGA 骨肉瘤测序数据共表达结果分析显示，SALL2作为转录因子可能调控S100A4的可能，相关分析结果显示SALL2与S100A4 mRNA呈明显负相关,相关系数rho=-0.67,P＜0.05. 在RAFLSs中上调SALL2基因表达后，明显抑制了S100A4的表达，提示SALL2基因可能参与了S100A4

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