CD36介导羧甲基赖氨酸致斑块内泡沫细胞外迁受抑的机制研究

The formation and inhibited emigration of foam cells are the key events in malignant remodeling of artery plaques. Advanced glycation end products (AGEs) play an important role in the course of diabetes- accelerating atherosclerotic progression. Our in vitro and in vivo studies demonstrated that Nε-carboxymethyl-Lysine(CML), a key active component of AGEs, may inhibit the migration of foam cells by the mediation of scavenger receptor CD36. However, the mechanism is still not clear.So,the present study will firstly use radioligand receptor binding assay to observe the characteristics of CML/CD36 binding.Secondly,we will take CML intervention, receptor blocking and cell migration experiments to explore the role of CD36 in CML inhibiting emigration of foam cells.Thirdly, we will clarify the CML/CD36 signaling pathway by exciting or blocking the multiple key signal points. Lastly, with the help of modern fluorescent labeling methods and genetically engineered cell transplantation, the dynamic observation of apoE-/- mice microPET CML receptor imaging and zebrafish cell migration confocal 3D reconstruction will be performed to explore the temporal and spatial characteristics of foam cell emigration from plaques in diabetic atherosclerosis and related mechnisms. The ultimate aim of this project is to illuminate the mechanisms of CD36 modulating foam cell emigration from plaques inhibited by CML which results in the progression of diabetic atherosclerosis. These series of experiments may provid a wonderful evidence for therapeutic strategy targeting mechanisms in future.

泡沫细胞的形成及外迁受抑是动脉壁斑块恶性重塑的关键环节，糖基化终末产物（AGEs）则是糖尿病加速动脉粥样硬化进展的重要推手。申请者前期研究显示AGEs关键活性成分羧甲基赖氨酸（CML）可能通过清道夫受体CD36介导而抑制泡沫细胞迁移，但机制尚不明确。本课题拟通过放射性配体受体结合实验观察CML与CD36的结合特性；通过CML干预、受体阻断及细胞迁移实验观察CD36在CML致泡沫细胞迁移受抑中的作用；通过多信号关键节点的激动与阻滞观察CML/CD36信号的传递路径；通过荧光标记及基因工程细胞移植并借助对apoE-/-小鼠microPET CML受体显像及对斑马鱼细胞迁移激光共聚焦三维重构的动态观察，探索糖尿病动脉粥样硬化斑块内泡沫细胞外迁的时空特征及相关机制，最终阐明CD36介导CML致斑块内泡沫细胞外迁受抑进而启动糖尿病动脉粥样硬化恶性演进的机制，为今后靶向机制的治疗策略提供新的切入点。

泡沫细胞的形成及外迁受抑是动脉壁斑块恶性重塑的关键环节，已有研究显示晚期糖基化终末产物（CML）的关键活性成分羧甲基赖氨酸（CML）可能通过清道夫受体CD36 介导而抑制泡沫细胞迁移，但机制尚不明确。本研究探讨了CD36在羧甲基赖氨酸致斑块内泡沫细胞外迁受抑中的作用和机制。研究发现：羧甲基赖氨酸（CML）可以抑制泡沫细胞的迁移，而在使用CD36中和性抗体进行干预后，主动脉斑块内泡沫细胞显著减少并大量迁移至主动脉旁淋巴结，提示CML可以通过CD36而影响泡沫细胞迁移能力。在细胞水平利用活性氧抑制与清除、胆固醇外流调控、激酶活性上调与下抑等手段进一步探讨其中潜在机制，发现CML/CD36信号通路可能是通过活性氧-FAK-Arp2/3-F-actin来影响细胞迁移的。同时我们使用PI3K/Akt信号激动剂740-YP干预 CML致泡沫细胞迁移受抑的过程，发现740-YP可以部分逆转该过程，这一结果表明PI3K/Akt通路参与了在CML致泡沫细胞迁移受抑的过程。另外我们合成了新型的18F-CML探针，发现其能较好的示踪斑块。在对糖尿病足截肢患者胫前动脉粥样硬化斑块内钙化的分析中，我们发现CML有促进钙化的作用，通过体外构建细胞钙化模型，揭示了CML/RAGE-ROS-p38MAPK- cbfα1-ALP钙化级联信号的传递途径。该研究拓展了对斑块内泡沫细胞迁移和斑块内钙化的分子机理认识，为抗动脉粥样硬化进展提供了一个新的靶点。