SAA通过TLR4信号通路促进类风湿关节炎血管新生的分子机制研究

发布时间：2018-09-18 16:31

【摘要】：目的类风湿性关节炎(RA)是一种慢性炎症性疾病,其发病特点主要为对称性的多关节滑膜炎和大量新生血管的形成。已有研究结果显示血清淀粉样蛋白A(SAA)在其发病机制中发挥重要作用。本研究首先分析了TLR4在RA患者滑膜组织中的表达,并进一步检测了SAA与TLR4的相互作用对HUVECs产生促血管生成因子VEGF,Ang-1,Ang-2的影响。最后,通过体外构建三维血管模型,深入探讨SAA与TLR4结合介导的NFκB信号途径在RA新生血管形成机制中的作用。本研究旨在探究SAA促进RA血管新生的分子机制,为RA的临床诊断和治疗寻找新的可靠的靶点奠定实验基础。方法1.免疫组织化学技术检测RA和OA患者滑膜组织中TLR4的表达。2.采用HUVECs体外细胞培养模型,细胞迁移实验和MTT实验分析SAA与TLR4的相互作用对内皮细胞迁移、增殖的影响。3.通过体外构建三维血管模型,分析SAA与TLR4的相互作用对内皮细胞形成新生血管的影响。4.ELISA方法分析SAA与TLR4的相互作用对内皮细胞产生促血管生成因子的影响。5.采用western blot方法分析在人脐静脉内皮细胞(HUVECs)中SAA-TLR4相互作用对NFκB信号途径活化的影响。结果1.免疫组化结果显示,TLR4广泛表达于RA滑膜组织的衬里层及衬里下层,主要表达在血管内皮细胞、成纤维细胞、巨噬细胞及血管周围;而在OA则少量表达于滑膜衬里层及成纤维细胞,且二者相比表达的量和位置均有显著差异。2.细胞迁移、MTT实验以及体外三维血管模型结果分别显示,SAA可显著促进HUVECs的增殖和迁移,以及相互连接构成新生血管;在TLR4被抑制后,SAA介导的内皮细胞的迁移、增殖程度以及血管新生明显受到抑制。3.ELISA结果显示SAA作用下HUVECs可产生VEGF、Ang-1、Ang-2等促进新生血管形成的因子,而TLR4被抑制后,VEGF的生成明显减少,其他几种因子所受影响不明显。4.Western blot实验结果显示HUVECs受SAA刺激后,NFκB分子明显活化,而一旦TLR4被抑制,NFκB的活化受到明显抑制。5.细胞迁移实验以及体外三维血管模型结果分别显示,在SAA作用下,HUVECs发生明显的迁移,并且相互连接构成新生血管;上述结果在NFκB被抑制后,明显受到抑制。结论1.TLR4在RA滑膜组织中呈高表达,且TLR4表达的位置和量与OA组相比均有显著差异,提示TLR4可能参与了RA的炎症反应和关节损伤。2.阻断TLR4的作用能够抑制SAA诱导的内皮细胞的增殖、迁移和新生血管形成,以及抑制内皮细胞产生促进血管新生的效应分子,提示SAA-TLR4的相互作用在血管新生中发挥重要作用。3.SAA-TLR4-NFκB信号通路可能直接参与了RA血管新生-血管翳的形成,进而造成RA患者关节软骨及骨的破坏。
[Abstract]:Objective Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by symmetrical polyarticular synovitis and angiogenesis. It has been shown that serum amyloid A (SAA) plays an important role in the pathogenesis of amyloid. In this study, we analyzed the expression of TLR4 in synovial tissue of RA patients, and further examined the effect of the interaction between SAA and TLR4 on HUVECs angiogenic factor VEGF,Ang-1,Ang-2. Finally, a three-dimensional vascular model was constructed in vitro to investigate the role of NF 魏 B signaling pathway mediated by SAA and TLR4 in the angiogenesis of RA. The purpose of this study was to explore the molecular mechanism of SAA promoting angiogenesis of RA and to find new and reliable targets for the clinical diagnosis and treatment of RA. Method 1. Immunohistochemical technique was used to detect the expression of TLR4 in synovial tissues of patients with RA and OA. HUVECs cell culture model, cell migration assay and MTT assay were used to analyze the effect of SAA and TLR4 interaction on endothelial cell migration and proliferation. Three-dimensional vascular model was established in vitro to analyze the effect of SAA / TLR4 interaction on endothelial cell neovascularization. 4. Elisa method was used to analyze the effect of SAA / TLR4 interaction on the production of angiogenic factors in endothelial cells. The effect of SAA-TLR4 interaction on the activation of NF 魏 B signaling pathway in human umbilical vein endothelial cells (HUVECs) was analyzed by western blot method. Result 1. Immunohistochemical results showed that TLR4 was widely expressed in the lining and sublining of RA synovial tissue, mainly in vascular endothelial cells, fibroblasts, macrophages and perivascular. However, in OA there was a small amount of expression in synovial lining layer and fibroblasts, and there was significant difference in the amount and position of expression between the two groups. The results of cell migration assay and three-dimensional vascular model in vitro showed that HUVECs could significantly promote the proliferation and migration of HUVECs, and interconnect with each other to form neovascularization, and after TLR4 was inhibited, SAA-mediated endothelial cell migration was induced. Proliferation and angiogenesis were significantly inhibited by Elisa. 3. The results of Elisa showed that HUVECs induced by SAA could produce VEGF,Ang-1,Ang-2 and other factors that could promote angiogenesis, while the production of TLR4 was significantly decreased after TLR4 was inhibited. The effects of other factors were not obvious. 4. The results of Western blot assay showed that HUVECs was significantly activated after SAA stimulation, but once TLR4 was inhibited, the activation of NF 魏 B was significantly inhibited. The results of cell migration experiment and three-dimensional vascular model in vitro showed that HUVECs migrated obviously and connected with each other to form neovascularization under the action of SAA, and the above results were obviously inhibited after NF 魏 B was inhibited. Conclusion there is a high expression of 1.TLR4 in RA synovium, and the location and quantity of TLR4 expression are significantly different from those in OA group, suggesting that TLR4 may be involved in the inflammatory reaction of RA and the injury of joint. 2. Blocking the effects of TLR4 can inhibit the proliferation, migration and angiogenesis of endothelial cells induced by SAA, and inhibit the production of angiogenic effector molecules by endothelial cells. The results suggest that the interaction of SAA-TLR4 may play an important role in angiogenesis. 3. SAA-TLR4-NF 魏 B signaling pathway may be directly involved in the formation of neovascularization and pannus of RA, which may lead to the destruction of articular cartilage and bone in RA patients.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.22

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