Nogo-B上调Tregs及其IL-10的产生在ALI小鼠肺保护中的作用及其机制研究

The endogenous anti-inflammatory effects of Tregs and IL-10 have attracted much attention in recent years. Our preliminary study has proved that Nogo-B played a protective role in the development and resolution of Acute lung injury (ALI), which may be involved in the regulation of biological functions of Tregs. In this study, we will employ wild type, Nogo-B-/- and IL-10-/- mice, as well as Tregs, CD4+T cells, macrophages and neutrophils as main objects. Using adenovirus to overexpress Nogo-B, neutralizing antibody to deplete endogenous IL-10 and specific T cell adoptive transfer techniques, we mainly observe the effect of Nogo-B expression on Treg cell quantity and IL-10 levels in ALI mice model. Meanwhile, we will clarify the functions and status of Tregs and its IL-10 synthesis in the protective role of Nogo-B within ALI mice. Then, with in vitro experiments, we further explore the potential underlying mechanism by which Nogo-B regulates Treg and its IL-10 production. Overall, we aim to clarify whether the protective role of Nogo-B in mice against ALI is achieved by regulating Treg and its synthesis level of IL-10, which not only help to elucidate the pathogenesis of ALI/ARDS but also provide new idea for seeking effective target for biotherapy.

近年来Tregs及IL-10所具有的内源性抑炎作用备受关注。我们前期研究证实：Nogo-B在ALI的发生、发展中具有重要的保护作用，其机制可能与其调控Tregs生物学功能有关。本研究拟以野生型、Nogo-B-/-和IL-10-/-小鼠ALI模型以及Tregs、CD4+T细胞、巨噬细胞、中性粒细胞为主要研究对象，分别采用腺病毒转染高表达、特异性中和抗体干预、T细胞过继转移等技术，观察调控Nogo-B表达对ALI小鼠体内Tregs数量及IL-10水平的影响；明确Tregs及其产生的IL-10在Nogo-B ALI小鼠肺保护中的作用及地位；同时采用体外实验探讨Nogo-B调控Tregs数量及其合成、分泌IL-10的可能机制，力求证明Nogo-B对ALI小鼠的肺保护作用主要是通过调控Tregs及其IL-10的产生来实现的。为进一步阐明ALI/ARDS的发病机制，寻求有效的生物治疗靶点提供新的思路。

急性肺损伤(ALI)/急性呼吸窘迫综合征(ARDS)是一种常见的呼吸系统危重症，临床表现为双肺弥漫性浸润及顽固性低氧血症。由于该病发病机制及病理生理过程不明, 临床缺乏药物及分子治疗手段。本课题组前期结果发现Nogo-B在ALI/ARDS的发生发展过程中发挥保护作用，但具体机制不明。近年来研究表明Treg细胞在ALI小鼠肺部炎症及损伤修复中扮演了重要功能。为进一步阐明Nogo-B在ALI中的保护作用及具体机制，本研究首先采用腺病毒转染及基因敲除技术，以LPS鼻腔滴注复制ALI小鼠模型，发现与对照组小鼠相比，Nogo-B高表达(Ad-Nogo-B)小鼠肺部炎症损伤减轻，修复能力增强，肺内 Treg 细胞数量及气道IL-10水平均有所上调。其次，以Tregs中和抗体预处理小鼠使其数量明显降低，发现Ad-Nogo-B的保护作用有所减弱；而采用 IL-10-/-小鼠亦得出与之类似的结论；表明Nogo-B对ALI小鼠的肺保护效应有赖于Treg细胞及其IL-10表达。此外，课题组在小鼠肺部鉴定出一种Fth1hi中性粒细胞表型，并且证明IL-10对ALI肺部炎症损伤的调节与其对该类中性粒细胞的调控密切相关。最后，体外研究结果得出，Nogo-B促进Treg细胞合成、分泌IL-10，而IL-10又能反向调控Treg细胞增殖等功能，这一正反馈调节通路可能参与了Nogo-B对ALI小鼠的保护作用。上述实验结果进一步阐明了ALI/ARDS的发病机制，为寻求有效的生物治疗靶点提供新的思路。