调节性T细胞对ApoE-/-小鼠腹主动脉瘤的保护作用及其机制研究

Abdominal aortic aneurysm (AAA) is a chronic vascular degenerative disease associated with a high mortality among elderly people. It is characterized by the deterioration of the aortic wall architecture leading to progressive segmental dilation with a lethal risk of aortic rupture. Unfortunately, the pathogenesis of AAA remains poorly understood and the therapeutic approach is limited to surgical repair. However, surgery is not indicated in asymptomatic patients or those with a small AAA or contraindications for surgery. Thus, a safe and effective pharmacological treatment that can prevent AAA formation and progression is highly warranted. AAA shares many characteristics of autoimmune diseases, the role of the immune system in aneurysmal disease has received considerable attention. However, the exact immunomodulatory mechanisms are obscure. CD4+CD25+ regulatory T cells (Tregs), a specific subpopulation of T cells, play a key role in maintaining immunological tolerance and control the progression of autoimmune diseases. Deficiency or dysfunction of Tregs disrupts immune homeostasis and leads to many pathological conditions.10 The forkhead/winged helix transcription factor (Foxp3), a specific marker of Tregs, is required for the maturation and function of Tregs as deficiency in Foxp3 results in dysfunction of Tregs. Recently, it has been reported that Foxp3 expression was reduced in peripheral CD4+CD25+ Tregs and the frequency of CD4+CD25+Foxp3 T cells was decreased in patients with AAA, suggesting that an impaired immunoregulation by Tregs may contribute to AAA development. However, it remains unknown whether Tregs treatment is able to prevent angiotensin II-induced AAA and if it is, what mechanismsms may underlie this therapic approach. Angiotensin II-mediated progression of AAA and animal models of AAA have been investigated with apolipoprotein E knockout (ApoE-/-) mice. The role of the immune system in AAA formation has received consideration. However, no studies have elucidated the effect of Tregs on the development of AAA. The present study was undertaken to test the hypothesis that adoptive transfer of Tregs may dose-dependently prevent angiotensin II-induced AAA in ApoE-/- mice, and explore the inherent molecular mechanisms of Tregs-mediated beneficial effects on AAA. A series of in vitro and in vivo experiments were performed to validate this hypothesis. Tregs treatment may provide a novel approach to the prevention of AAA.

为了明确调节性T细胞（Tregs）是否可在腹主动脉瘤（AAA）形成中发挥有益作用及其具体的作用机制，本课题首先在高脂喂养的ApoE敲基因小鼠泵入Ang II，观察外源性给予Tregs、Tregs体内抑制及FoxP3敲除后对AAA的发生率和分型的影响，并利用激光共聚焦、免疫组化、实时定量RT-PCR、Western blot等技术对小鼠AAA组织细胞凋亡、胶原代谢、氧化应激、炎症反应等指标和关键信号分子进行定性定量分析。体外试验部分Tregs与血管平滑肌细胞或巨噬细胞共培养后给予Ang II刺激，观察其对细胞凋亡、胶原合成和降解、氧化应激、炎性反应以及下游信号通路的影响，明确Tregs对血管平滑肌细胞和巨噬细胞的作用及其分子机制。本课题的实施将证实我们的假说"外源性输入的Tregs可抑制AAA的形成"，并阐明相关的分子机制，从而为预防和治疗AAA这一重大疾病提供新的治疗策略。

验证在血管紧张素 II诱导的ApoE-/-小鼠腹主动脉瘤（AAA）模型中，调节性T细胞（Tregs）是否可以剂量依赖性的抑制腹主动脉瘤的形成。首先在高脂喂养的ApoE-/-小鼠中，通过持续泵入血管紧张素 II 28天建立AAA模型，后随机分为PBS组，小剂量Tregs组，大剂量Tregs组和PC干预组（拮抗体内Tregs）。AAA的发生率分别为80%，76%，27%和71%。Tregs明显抑制炎症细胞（巨噬细胞和CD4+ T细胞）的浸润，炎症因子（MCP-1，IL-6和ICAM-1）和基质金属蛋白酶（MMP-2和MMP-9）的表达及氧化应激反应，减少血管平滑肌细胞的凋亡，促进抗炎因子（IL-10和TGF-β）的表达。体外研究中， 通过对Tregs和人主动脉平滑肌细胞共培养后证实了Tregs可以减少炎症因子、MMP-2、MMP-9和凋亡相关的Fas-L的产生，抑制NF-κB和ERK1/2的活化，并促进抗炎因子IL-10和TGF-β的表达。另外，Tregs促进巨噬细胞表型由M1型向M2型的转变。应用Transwell板和特异性IL-10及TGF-β的中和抗体，则部分逆转了Tregs的相关作用。因此，外源性Tregs可剂量依赖性的降低血管紧张素 II诱导小鼠AAA的发生率和严重性，其机制包括抑制血管局部炎症细胞的浸润，炎症因子和基质金属蛋白酶的表达及氧化应激反应，减少血管平滑肌细胞的凋亡，促进抗炎因子的表达，而且证明Tregs是通过细胞-细胞直接接触和分泌特异性的因子（IL-10和TGF-β）来发挥作用的。