免疫干预转变巨噬细胞胆固醇代谢通路抑制动脉粥样硬化研究

The general process of pathogenesis of atherosclerosis involved macrophage is initiated by the phagocytosis of oxidized lipid via pattern-recognition receptors, which can induce and maintain the inflammatory response and, ultimately, the macrophage will die through apoptotic or necrotic mechanism after lipid overloaded. One strategy to prevent the pathogenesis of atherosclerosis is to transform the macrophage phagocytosis pattern to the oxidized lipid and promote to efflux the cholesterol from atherosclerosis plaque. We hypothesize that using the specific antibody against oxidized lipid will induce the macrophage in dealing with lipid from "phagocytosis via pattern recognition receptors-inducing inflammation" to "phagocytosis via specific antibody-promoting cholesterol efflux". This pathway of cholesterol metabolism will inhibit inflammation reaction, promote cholesterol efflux, avoid excessive lipid load, and prevent macrophage death fate, which all contribute to the pathogenesis of atherosclerosis. The main parts in the present project include: the intervention with anti-MDA-apoB-PF antibody in apoE-/- mouse model, the observation of cholesterol phagocytosis and metabolic changes in animal tissue; the observation of the inhibition of NF-κB regulated inflammation reaction; the observation of the expression PPARα and cholesterol efflux related molecules; the observation of the fate of macrophage overloaded lipid, including autophagy, apoptosis, and necrosis, and at last we will observe the difference of atherosclerosis lesions in different treatment groups. Meanwhile, we will investigate the mechanisms in macrophage cells in vitro. The main purpose is to elucidate the role and mechanism of antibody against MDA-apoB-PF which mediates the transforming of cholesterol metabolism pathway in macrophage in the prevention and treatment of atherosclerosis.

巨噬细胞通过模式识别受体吞噬氧化脂质，启动维持炎症反应并最终凋亡坏死是动脉粥样硬化发展的基本脉络。如能利用特异性抗体分子作为介导，免疫干预转变这一通路，使巨噬细胞对脂质的"模式识别受体非特异性吞噬-诱导炎症反应"处理模式转变为"特异性受体吞噬-胆固醇外流转运"模式，可抑制炎症反应形成，促进斑块内胆固醇外流，避免脂质过度负荷，减轻巨噬细胞死亡命运，实现动脉粥样硬化的抑制。具体内容包括：制备负载MDA-apoB-PF抗体的apoE敲除鼠模型，观察血管组织巨噬细胞胆固醇吞噬和代谢途径的改变，NF-κB炎症系统的抑制，PPARα及胆固醇外流分子表达的增强；观察负脂巨噬细胞自噬、凋亡的命运差异和动脉粥样硬化病变的形成情况。同时，从离体细胞水平探讨上述作用及其机制，并用多层次的药物干预作为上述作用环节的验证。最终明确MDA-apoB-PF抗体介导巨噬细胞胆固醇代谢通路的转变对动脉粥样硬化的防治作用。

本课题旨在“通过免疫干预”转变“巨噬细胞胆固醇代谢通路”抑制动脉粥样硬化，其核心内容为利用特异性抗体分子作为介导，免疫干预转变这一通路，使巨噬细胞对脂质的“模式识别受体介的非特异性吞噬-诱导炎症反应”处理模式转变为“特异性受体介导的免疫吞噬-胆固醇外流转运”模式，从而抑制炎症反应形成，促进斑块内巨噬细胞胆固醇外流，避免脂质过度负荷及巨噬细胞泡沫化，实现动脉粥样硬化的抑制。本研究原计划利用四年时间采用动物模型及细胞实验验证免疫干预对胆固醇代谢通路的转变及抗动脉粥样硬化的效应，但最终仅获得一年期的小额资助，我们在有限的一年时间内仅对部分内容进行了研究，主要取得的研究成果包括：（1）制备并提纯针对氧化apoB100三片段多肽的特异性抗体。（2）在细胞水平验证氧化apoB100片段特异性抗体可显著减少巨噬细胞脂质负荷并显著增加巨噬细胞的脂质外流。（3）在细胞水平，氧化apoB100片段特异性抗体可显著抑制巨噬细胞的NF-κB炎症通路，促进PPARα介导的脂质外流代谢通路，同时可增加胆固醇酯合成关键酶ACAT的表达，且以上效应与抗体的滴度呈正相关。从以上研究可以明确，氧化apoB100多肽片段特异性抗体可以抑制巨噬细胞对脂质的炎症吞噬通路，增强脂质代谢及外流通路，最终起到转变胆固醇代谢通路，减轻巨噬细胞脂质沉积及动脉粥样硬化形成的作用。一年的研究虽然仅限于离体研究，但这是整个研究的重要基础和核心组成部分，达到了此年度可能实现的研究目标。