节律因子Clock调控肥大细胞脱颗粒参与动脉粥样硬化斑块破裂的研究

Atherosclerotic plaque rupture is the most important cause of acute cardiovascular events, which often burst in the morning. Mast cells (MCs) play a key role in plaque rupture, and MCs’ degranulation is related with plaque instability. Data from our preliminary studies have showed that expression of core circadian gene Clock is closely associated with acute coronary syndrome (ACS) and the release of catecholamine could promote MC degranulation. Based on these, a novel mechanism is proposed “Clock gene regulates catecholamine and activates MCs, which would accelerate the pathological process of plaque rupture”. In order to prove it, carotid vulnerable plaque model are inducing in ApoE knockout mice, of which the expression of Clock and FcƹRI during 24-hour period are monitored with bioluminescence imaging. Then, mechanisms of Clock regulating catecholamine and stimulating FcƹRI expression are investigated with lentivirus in MC and Clock/ApoE knockout mice. Our study would not only help to uncover the potential mechanisms of plaque rupture, but also provide new methods and targets for clinical intervention.

动脉粥样硬化斑块破裂是急性心血管事件发生的最主要病因，具有突发性的临床特点和“晨起高峰”的节律现象。在斑块突然破裂中扮演重要角色的肥大细胞（MC），其激活脱颗粒和斑块不稳定性增加有关。我们前期研究发现：节律因子Clock与急性冠脉综合征（ACS）发病密切相关，且晨起儿茶酚胺的释放能促进MC脱颗粒。为此，提出“Clock可通过调控儿茶酚胺对MC的激活，进而诱发斑块破裂”的假说。我们首先在ApoE基因敲除小鼠上构建颈动脉易损斑块模型，通过活体荧光成像技术，观察Clock和MC表面受体FcƹRI的昼夜表达谱，并分析其与斑块稳定性之间的关系；随后，在MC和Clock/ApoE双基因敲除小鼠动物模型两个层次上采用慢病毒等手段，探索Clock通过调节儿茶酚胺刺激MC表面受体FcƹRI的表达，参与斑块破裂的分子机制。深入的研究不但有助于揭示诱发斑块破裂的新机制，而且为今后临床干预治疗提供新方法和新靶点。

动脉粥样硬化斑块破裂继发血栓形成阻塞管腔是急性心血管事件发生的最主要病因，多发于晨醒后（“晨起高峰”现象），呈现出明显的昼夜节律性。但节律是否可调节斑块的破裂及其病理病机仍不清楚。.我们首先分析了急性冠脉综合征（acute coronary syndrome，ACS）患者12种主要节律因子的表达变化谱，筛选AS斑块破裂可能的致病节律基因，结果提示：相较于正常对照组，ACS患者外周血单个核细胞Clock基因表达呈现明显的节律变化，且高峰位于6:00AM。随后对节律基因Clock参与动脉粥样硬化斑块破裂的分子机制进行研究，体内研究选用apoE-/-小鼠作为小鼠动脉粥样硬化模型，体外研究选择小鼠主动脉内皮细胞（MAECs），证实了：小鼠模型中CLOCK、白血病抑制因子（LIF），细胞间黏附分子1（ICAM-1），perilipin 2（ADFP），核因子κB（NF-κB）和PAI-1的水平以及动脉粥样硬化斑块的数目均明显增加。染色质免疫共沉淀（CHIP）显示CLOCK基因直接与PAI-1的启动子区域结合，CLOCK基因能够正向调控LIF，ICAM-1，ADFP，NF-κB和PAI- 1。CLOCK表达的降低会下调VCAM-1，CCL-2和CCL-5的表达，以及粘附细胞和动脉粥样硬化斑块的数量，MAECs中上调PAI-1表达伴随Clock表达同步上调，诱发动脉粥样硬化斑块形成。深入的研究不但有助于揭示诱发斑块破裂的新机制，而且会为今后临床干预节律紊乱治疗性药物的筛选和开发提供新方法和新靶点。