热中性温度调节肥大细胞外泌体分泌和 FcεRI表达的作用及分子机制

Allergic asthma is one of the most severe conditions compromising human health and quality of life. IgE is the main mediator of allergic reactions,and effectively inhibiting or neutralizing IgE can suppress the development of allergic reactions.We first reported that IgE level in serum was significantly lowererd in asthmatic mice at the ambient temperature of +30℃, which is the thermoneutral temperature for mice , as compared to that of +20℃. However,the underlying mechanism remains to be elucidated. It has been reported that mast cell-derived exosomes in mice can neutralize IgE, thus reducing the activation of mast cells and creating an anti-IgE effect. Furthermore, p53 promotes exosome secretion under thermal stress in multiple cancer cell lines. Our previous findings showed that p53 can be activated in mast cells by thermal stimulation, and the expression of FcεRI is much less in p53-/- mast cell-derived exosomes. Therefore, we propose that the reduction of serum IgE level in asthmatic mice could be related to the activation of p53 by thermoneutral temperature, which promotes the expression of FcεRI and secretion of exosomes in mast cells. This study aims to investigate the mechanism of p53 regulating exosomes secretion and FcεRI expression of mast cells in thermoneutral environment with p53 knockout mice and animal cell and molecular biology techniques. Our study will further the understanding of pathogenesis to asthma, and provide a novel therapeutic target for its treatment.

过敏性哮喘已成为影响人类健康和生活质量的最严重疾病之一。IgE是过敏反应的主要介质，有效抑制或中和IgE，能够抑制过敏反应的发生发展。我们在国际上首次报道与20℃相比，30℃（热中性温度）中哮喘小鼠血清IgE水平降低，但机制尚不清楚。研究表明小鼠肥大细胞外泌体可结合IgE，抑制肥大细胞激活，出现肥大细胞外泌体抗IgE效应。另有报道热刺激肿瘤细胞可激活p53促进外泌体分泌。我们前期研究发现热刺激肥大细胞可激活p53，p53-/-小鼠肥大细胞外泌体FcεRI表达减少。因此我们推测，热中性温度下哮喘小鼠血清IgE水平降低可能与热中性温度激活p53，进而使肥大细胞FcεRI表达和外泌体分泌增加有关。本课题拟运用p53基因敲除小鼠，通过动物细胞和分子生物学技术，研究热中性温度调控肥大细胞外泌体分泌和FcεRI表达的作用和机制。该研究将加深对哮喘发病机制的新认识，为寻找哮喘的治疗新靶点提供科学依据。

背景：热疗是治疗气道炎症性疾病（包括过敏性哮喘）的一种方法。我们前期报道了哮喘小鼠血清IgE浓度随着环境温度升高而降低，但其机制不明。本研究旨在研究温度变化是否通过影响肥大细胞（Mast cells，MC）来源的外泌体及外泌体表面IgE高亲和受体 （FcεRI），进而介导依赖MC激活和卵清蛋白诱导的实验性哮喘。.方法：将小鼠骨髓来源的MCs和两种肥大细胞系（RBL-2H3和P815），在29℃、37℃和41℃温度下培养2小时，从细胞上清液分离出外泌体。评估温度对RBL-2H3衍生的外泌体体及FcεRI，对MC激活和实验性哮喘的影响。通过RNA-seq筛选温度相关的影响外泌体分泌的关键基因，并通过siRNA敲低进行验证。.结果:较高的温度能促进MC衍生外泌体的分泌效率，且上调了外泌体中FcεRI的表达。体外实验发现较高温度下产生的外泌体能够结合游离IgE，竞争性地抑制MC与游离IgE的结合，来防止MC激活。41℃培养的RBL-2H3源性外泌体通过腹腔注射在哮喘小鼠体内，发现与其他两个温度相比，更能够缓解哮喘小鼠的症状。RBL-2H3细胞中Rab3a的表达随着温度升高而上调。在RBL-2H3来源的外泌体中，Rab3a的敲除抑制了外泌体的分泌效率和FcεRI的表达，但在RBL-2H3细胞中的FcεRI无变化。.结论:MC衍生的外泌体通过Rab3a依赖的外泌体分泌和上调外泌体中FcεRI的表达，以温度敏感的方式抑制MC激活和实验性哮喘。因此，热修饰的外泌体具有潜在的治疗应用。