高血脂强炎症反应ApoE/STAT4双基因敲除小鼠动脉硬化易感模型建立及其发病机制研究

Atherosclerosis (As) and vulnerable plaque rupture plays key roles in acute coronary events, but its pathogenesis remains to be clarified. Role of hypercholesterolemia and enhanced inflammatory reaction in atherogenesis needs to be further studied. ApoE knockout mice (ApoE KO) are widely used in As research, but for pathological relationship of vulnerable plaques and As later stage are relatively limited. A novel atherosclerosis susceptibility murine model with hypercholesterolemia and enhanced inflammatory reaction will help to elucidate the role of lipid metabolism and inflammation abnormality in atherogenesis and in the development of vulnerable plaque. In previous study, we found that histamine and STAT4 signal genetic deficiency can promote myeloid immune cell proliferation, differentiation and mobilization. ApoE/STAT4 double knockout mice (ApoE/STAT4 DKO) are more susceptible to develop a large plaque compared to ApoE KO mice with HFD diet. Thus, we propose the hypothesis: we can establish a novel atherosclerosis susceptibility murine model with hypercholesterolemia and enhanced inflammatory reaction by ApoE/STAT4 DKO mice. This mouse model may finally develop a vulnerable plaque with histamine or angiotensin induction. Targeted histamine-JAK/STAT4 signal will help to investigate the mechanisms invloved in the expression of CD11b+Gr-1+ immature myeloid cells and macrophage polarization in atherogenesis. The establishment of atherosclerosis susceptibility murine model will help to clarify the pathogenesis of As, provides a reliable research tool for drug screening, prevention and treatment of As.

动脉粥样硬化（As）易损斑块的发展和破裂在急性冠脉事件中起着关键的作用，但其发生机制有待阐明。脂代谢异常和炎症反应在As发生中的作用尚不明确。ApoEKO小鼠广泛应用于As相关研究，但用于研究易损斑块与As末期的病理关系作用却比较局限。一种既有高血脂，又有增强炎症反应的As敏感小鼠模型的建立，将有助于阐明血脂代谢和炎症反应异常对As易损斑块发展的作用，以及二者之间的相互“作用”。在前期研究中，我们发现组胺或STAT4信号缺失可以引起髓系免疫细胞的增殖、分化和动员的异常，ApoE/STAT4双基因敲除小鼠较ApoEKO小鼠更易发生大的斑块。因而我们设想： ApoE/STAT4双基因敲除小鼠因为具备ApoE KO小鼠的高血脂和STAT4KO小鼠的异常免疫细胞动员，有利于建立一种新型、有易损斑块的As易感小鼠模型。该模型的建立，有利于阐明As发病机制，为筛选防治As的药物提供可靠的研究工具。

炎症反应和免疫细胞在动脉粥样硬化（As）发生发展中的作用及机制仍不明确。本项目把ApoE KO小鼠的高血脂高胆固醇和STAT4 KO和STAT6 KO小鼠的异常免疫细胞反应结合起来，建立了ApoE/STAT4和ApoE/STAT6两种双基因敲除小鼠，研究炎症与免疫细胞相关的As发病机制。研究发现：（1）阻抑STAT4信号，显著促进高脂饲养ApoE/STAT4 DKO小鼠As斑块的发生，而阻抑STAT6信号，却可以抑制ApoE/STAT6 DKO小鼠As斑块的发展； ApoE/STAT4 DKO与对照ApoE KO小鼠比较，AS斑块具有更大的脂质核心，更薄的纤维帽。（2）阻断STAT4或STAT6信号，都不显著改变ApoE/STAT4 DKO和ApoE/STAT6 DKO小鼠高血脂水平。（3）STAT4KO促进CD11b+IMCs向巨噬细胞分化和泡沫细胞转化；可下调PI3K/Akt signaling 和上调ACAT-1酶活性，促进巨噬细胞内胆固醇的蓄积。（4）应用HDCKO和STAT6KO小鼠，实验证明histamine/HR/STAT6 signal介导了histamine对CD11b+Ly6Chigh 巨噬细胞分化的调控。（5）ApoE/STAT4 DKO和ApoE/STAT6 DKO小鼠和骨髓分离细胞进行DC诱导培养，初步发现STAT4和STAT6敲除对DC分化没有显著影响，但是它们对DC成熟有相反的效用。（6）对ApoE和STAT4/ApoE DKO小鼠的主动脉血管和CD11b+细胞诱导的巨噬细胞进行蛋白质组学分析，获得了数十个差异表达蛋白。（7）以CD11b+细胞为靶标，建立一种新型无创、在体监测易损斑块和肿瘤的分子影像技术。通过本项目的实施，我们构建了ApoE/STAT4 和ApoE/STAT6两种与As有关敲除小鼠模型，丰富了As研究中炎症与免疫细胞相关机制的动物模型。既往STAT4/STAT6信号的研究，多集中在它们对T淋巴细胞分化和功能的影响。本项目主要从STAT4/STAT6信号对髓系细胞，尤其是针对巨噬细胞和树突状细胞的分化和成熟，开展相关研究。研究成果将为阐明先天免疫在As发生中的作用及机制提供新的证据，为防治As提供新的药物靶标。本研究实施期间，已发表标注有项目编号的SCI收录英文文章4篇（通讯作者3篇，第一标注2篇），中文核心期刊文章1篇