利用CRISPR/Cas9技术构建D374Y-PCSK9转基因兔动脉粥样硬化易损斑块模型

Vulnerable plaque formation and rupture is the major cause of clinical acute cardio-cerebrovascular events. But, the progress on the understanding of its pathogenesis and the development of intervention measures are slow, which are predominantly due to the lack of ideal animal model. Previously, we developed a murine model of carotid atherosclerotic vulnerable plaque by activation of renin-angiotensin system and induction of local shear stress simultaneously in ApoE-/- mice. Histological analyses indicated that all left common carotid artery lesions had vulnerable features and 50% of the mice showed plaque rupture with a lumen thrombus. However, the significant differences of lipoprotein metabolism and plaque features between mice and human restrict its application. Based on our preliminary studies, we will firstly generate a rabbit model of spontaneous hypercholesterolemia by CRISPR/Cas9 mediated D374Y mutation of PCSK9. Furthermore, we intent to establish a model of vulnerable plaque by combined partial ligation of the left renal artery and left common carotid artery to induce activation of renin-angiotensin system and local shear stress respectively in D374Y-PCSK9 transgenic rabbit. In addition, we will investigate the changes of serum and intraplaque protein characteristics in the process of vulnerable plaque formation by proteomics. Finally, we strive to identify the biomarker of vulnerable plaque and the key proteins leading to vulnerable plaque formation. The overall purpose of this project is to provide new ideas and methods to identify and stabilize the existing atherosclerotic plaques and to prevent acute cardiac events.

易损斑块形成和破裂是导致临床急性心脑血管事件的主要原因，但其发病机制和干预措施远未完全阐明。我们以往采用全身性和局部性联合干预方法，在ApoE-/-小鼠成功构建了易损斑块模型，8周内易损斑块形成率100%，斑块破裂伴血栓形成率达50%。但小鼠脂蛋白代谢和动脉粥样硬化病变与人类差异较大，限制了其应用。因此本项目拟以新西兰兔为模式动物，利用CRISPR/Cas9技术构建D374Y-PCSK9转基因家兔，使其自发产生高脂血症，然后通过部分结扎左颈动脉和左肾动脉的方法，改变血管局部剪切力的同时激活全身内源性肾素-血管紧张素系统，拟建立一种易损斑块发生率高、实验周期短的家兔动脉易损斑块模型；同时运用蛋白组学技术，考察易损斑块形成过程中血清及斑块内组织蛋白质特征变化网络，筛选鉴定可用于诊断的特征性分子标记物和导致斑块不稳定的节点蛋白，为易损斑块的诊断和干预提供新思路。

近些年研究显示，放疗能够激活大多数肿瘤细胞自噬过程，而抑制自噬能够减少接受放射治疗的恶性肿瘤患者肿瘤细胞逃逸，是潜在的增强放射治疗疗效的重要手段，然而，抑制自噬是否会增加放疗相关心血管病风险，目前尚未可知。我们通过部分结扎ApoE-/-小鼠颈动脉联合局部X线放射的方法构建放射线相关的小鼠动脉粥样硬化模型，发现X线辐射能够显著增加小鼠颈动脉斑块面积、巨噬细胞比例和脂质沉积面积，降低平滑细胞比例、胶原含量。Western blotting和免疫荧光结果显示，X线辐射可显著降低血管局部P62和LC3II水平，减轻7-KC诱导的巨噬细胞P62和LC3II的上调，提示X线辐射促进巨噬细胞自噬流。阻断自噬可进一步增大小鼠颈动脉斑块面积，表明X线辐射诱导的自噬在动脉粥样硬化进展中发挥着保护作用。最后，我们揭示了X线辐射可上调炎症水平MCP-1、TNF-α、IL-1α炎症因子，P65核转位，表明X线激活NFκB炎症通路，而阻断自噬可进一步引起NFκB炎症通路的激活。综上所述，X线辐射加速小鼠颈动脉粥样硬化进展，同时促进斑块局部自噬流；自噬激活通过抑制炎症反应负向调控X线辐射诱导的斑块进展；抑制自噬进一步加重X线辐射诱导的动脉粥样硬化。因此，自噬抑制治疗有可能增加接受放疗的肿瘤患者心血管事件发生风险。