基于oxLDL/LOX-1/NF-κB途径诱导巨噬细胞TF表达及其干预的分子机制研究

Tissue factor (TF) expression induced by oxidized low-density lipoprotein (oxLDL) in monocyte-derived macrophages (MDMs) played a key role in atherosclerotic plaque rupture and thrombosis formation. However, oxLDL induction of TF expression in MDMs via NF-κB mediated by LOX-1 and its intervention remain undefined. Studies have shown that beta 2-Glycoprotein I (β2GP I）inhibit the pathogenicity of oxLDL through the combination of oxLDL. Then, we separated and identified the epitopes of oxLDL, 9-COOH-7-KC, which mediated the binding of β2GP I with oxLDL. After experimental analysis and molecular docking calculations, we preliminarily confirmed that binding of β2GP I DⅤ with oxLDL via 9-COOH-7-KC might be an important way to block the binding of oxLDL with LOX-1 mediated by 9-COOH-7-KC and thereby inhibit oxLDL induction of TF expression. Thus, we are going to do the following work by biological strategy of gain of function(GOF)and loss of function(LOF): First, we intends to prove TF expression in MDMs induced by this new oxLDL/LOX-1/NF-κB signaling pathways. Second, with the 9-COOH-7-KC as target, we intends to investigate the intervention effect、molecular mechanisms and interaction sites of blocking the binding of oxLDL with LOX-1 and thereby blocking oxLDL induction of TF expression by β2GP ⅠDⅤ.This study provides the theoretical and laboratory basis for the prevention and treatment of clinical atherothrombosis. ?????

oxLDL诱导的巨噬细胞TF表达对动脉粥样硬化斑块破裂及继发血栓形成具有重要作用，但基于LOX-1受体介导oxLDL经由NF-κB对巨噬细胞TF表达及其干预的研究尚不清楚。研究显示，β2GPⅠ通过与oxLDL结合阻抑了oxLDL的致病性。于是我们分离了介导二者结合的oxLDL表位结构9-COOH-7-KC, 经实验及分子对接分析显示，β2GPⅠ DⅤ经由9-COOH-7-KC与oxLDL的结合，是封闭9-COOH-7-KC介导的oxLDL与LOX-1结合进而阻断oxLDL诱导TF表达的重要途径。为此，本课题拟采用生物学功能获得和缺失策略，探明LOX-1介导oxLDL经由NF-κB诱导巨噬细胞TF表达这一全新信号通路；以9-COOH-7-KC为药物靶点，阐明β2GPⅠ DⅤ封闭oxLDL与LOX-1结合进而阻断其诱导TF表达的抑制效应、分子机制和作用位点，为动脉血栓的预防和治疗提供理论依据。

背景：. oxLDL诱导的巨噬细胞TF表达对动脉粥样硬化斑块破裂及继发血栓形成具有重要作用。巨噬细胞表面的清道夫受体血凝素样氧化低密度脂蛋白受体LOX-1，与oxLDL结合可促发信号转导，引发一系列动脉血栓病理变化。研究显示，β2GPⅠ通过与oxLDL结合阻抑了oxLDL的致病性。于是我们分离了介导二者结合的oxLDL表位结构9-COOH-7-KC, 经实验及分子对接分析显示，β2GPⅠ DⅤ经由9-COOH-7-KC与oxLDL的结合，是封闭9-COOH-7-KC介导的oxLDL与LOX-1结合进而阻断oxLDL诱导TF表达的重要途径。然而，LOX-1是否介导了oxLDL诱导的巨噬细胞TF表达以及β2GPⅠ DⅤ对其表达的抑制效应、分子机制尚未清楚。.内容：.1.检测oxLDL对巨噬细胞TF表达的调节及LOX-1受体对此途径的介导作用。.2.建立β2GPⅠ DⅤ的原核和表达体系，检测rβ2GPⅠ DⅤ对oxLDL诱导巨噬细胞TF表达的抑制作用.3.建立表达人LOX-1受体的细胞模型，利用此模型检测rβ2GPⅠ DⅤ对于阻断oxLDL与LOX-1结合进而阻断TF表达的作用。.结果：.1.低浓度oxLDL能够显著上调J774A.1细胞TF、LOX-1的mRNA和蛋白的表达水平。与scrambled siRNA相比，LOX-1 siRNA沉默组TF的蛋白表达量显著下调69%，说明LOX-1参与了oxLDL诱导的巨噬细胞TF表达。.2. 构建了β2GPⅠ DⅤ的表达系统，实现了β2GPⅠ DⅤ的体外表达，ELISA实验表明，P. rβ2GPⅠ DⅤ经由oxLDL上的阴性磷脂表位结构9-COOH-7-KC与oxLDL结合。.3. Western Blot证明P. rβ2GPⅠ DⅤ能够浓度依赖性抑制oxLDL诱导的TF和LOX-1上调表达，流式结果显示，P. rβ2GPⅠ DⅤ浓度依赖性的干预LOX-1与DiI-oxLDL的结合，因此表明P. rβ2-GPI DV通过抑制和干预细胞膜表面oxLDL与LOX-1的结合进而阻断了TF和LOX-1的上调表达。.科学意义：. 为最终防治TF导致的动脉粥样硬化斑块破裂和继发的血栓及今后的心血管事件的发生提供理论依据。