最近，我们针对动脉粥样硬化（AS）的两个关键病因，突破性的设计了一种含有热休克蛋白65（HSP65）和胆固醇酯转移蛋白（CETP）表位的重组蛋白HCPTC，经鼻粘膜免疫明显减轻AS，呈现出诱导免疫耐受和免疫反应的双重性以及既抗炎又调脂的双重性。但其机制不明。基于树突状细胞（DCs）在激发免疫反应和诱导免疫耐受方面的关键作用，本项目拟首先研究HCPTC对鼻粘膜DCs的表型、成熟度和功能的影响，以及HCPTC与DCs表面受体的亲和作用及DCs接触HCPTC后的表现，揭示HCPTC产生双重免疫反应的机制；其次，研究HCPTC对调节性T细胞（Treg）Foxp3基因甲基化修饰的影响及其作用机制，揭示HCPTC诱导免疫耐受的机制；再者，研究HCPTC对CETP活性和Treg数量和功能的影响，揭示HCPTC产生双重治疗作用的机制。本项目将为AS粘膜疫苗的设计以及AS的防治提供新的思路和方法。

Recently, an attractive recombinant protein HCPTC designed to contain heat shock protein 65 (HSP65) and cholesterol ester transfer protein (CETP) epitopes based on two key etiological factors of atherosclerosis (AS), significantly reduce AS by the nasal immunization, which show the dual abilities of inducing immune tolerance and immune response, as well as the dual effects of anti-inflammatory and lipid-modify. But the mechanisms remain to be unknown. Based on the key roles of dendritic cells (DCs) in stimulating immune response and inducing immune tolerance, the project intends to firstly study the effects of HCPTC on phenotype, maturity and functionality of nasal DCs, as well as affinity interaction between HCPTC and surface receptor of DCs, also the behavior of DCs after HCPTC is recognized by DCs, which will reveal the mechanisms of two kinds of immune reaction induced by HCPTC; secondly, to study the effects and mechanisms of HCPTC on the methylation of Foxp3 gene in regulatory T cells (Treg), which will reveal the mechanisms of immune tolerance induced by HCPTC; thirdly, to study the effects of HCPTC on CETP activity and Treg number and function, which will reveal the mechanisms of double therapeutic effects produced by HCPTC. The project will provide new ideas and methods for the design of mucosal vaccine in AS and the prevention and treatment of AS.

动脉粥样硬化（atherosclerosis，AS）是由多种复杂因素引起的慢性疾病，也是多种心血管疾病的发病基础，研究AS的防治药物意义重大。最近，我们针对动脉粥样硬化（AS）的两个关键病因（自身免疫、脂质紊乱），设计了一种含有热休克蛋白65（HSP65）和胆固醇酯转移蛋白（CETP）表位的重组蛋白HCPTC，经鼻粘膜免疫明显减轻AS，但其效应机制不明。基于树突状细胞（DCs）在激发免疫反应和诱导免疫耐受方面的关键作用，本项目首先研究HCPTC对鼻粘膜DCs的表型、成熟度和功能的影响，以及HCPTC与DCs表面受体的亲和作用及DCs接触HCPTC后的表现，揭示HCPTC产生双重免疫反应的机制；其次，研究HCPTC对调节性T细胞（Treg）Foxp3基因甲基化修饰的影响及其作用机制，揭示HCPTC诱导免疫耐受的机制；再者，研究HCPTC对CETP活性和Treg数量和功能的影响，揭示HCPTC产生双重治疗作用的机制。结果表明，鼻粘膜接受HCPTC抗原后，粘膜DCs表面的清道夫受体可以对HCPTC进行识别，粘膜部位的细胞因子微环境发生变化，DCs的成熟及活化程度下降，促使粘膜部位以低成熟度的DCs为主；随之变化的是DCs摄取HCPTC的能力下降，粘膜DCs向颈部淋巴结迁移的能力下调，DCs表面的共刺激分子信号强度减弱，导致其不能高效刺激T细胞活化。另外，鼻粘膜接受HCPTC抗原后，Treg细胞的DNMTs数量和酶活力下降，引起关键的Foxp3基因启动子和CNS2区域的甲基化水平下降；随之，Foxp3的表达上调，Treg细胞的数量及免疫抑制功能加强，而Th17数量下降，体内呈现抗炎细胞因子水平上调、促炎细胞因子水平下调的免疫调节状态。再者，鼻粘膜接受HCPTC抗原后，体内产生了针对CETP的抗体，随之，CETP数量和酶活力下降，最终HDL-C水平上调、LDL-C水平下调。由此可见，鼻粘膜接受HCPTC抗原后，机体粘膜DCs、Foxp3基因甲基化水平、免疫平衡和脂质平衡均发生了良性改变，最终导致动脉粥样硬化斑块程度明显减轻。本项目发现了促使粘膜疫苗针对不同抗原呈现多重免疫效应的原因和方法，为AS粘膜疫苗的设计、效应评价以及AS的防治提供了新的思路和方法。