不同细胞来源exosome-FcεRIα中和血清游离IgE降低过敏反应的作用及机制

Mast Cell(MC) is effector cell for hypersensitivity, and express high affinity IgE receptor FcεR I and stem cell factor receptor KIT ,which have synergistic effect, involved in its activation, differentiation and survival. Exosome is ideal carrier for target moleculars and drugs. Mast Cell exosome（MC-exo）, which contain intracellular particles, mRNAs, can shuttle from intercellular space by carrying FcεRI and KIT to deliver and release inflammatory mediators and cyotokines. FcεRIα chain is the binding site for IgE. Based on prvious studies about FcεRI on the function of MC，MC-exo and DC, we design experiments by harvest Bone Marrow Mast Cell(BMMC) from mice with allergic asthma and imDC from FcεRIα transgenic mice and get exosomes from these two cells. By delivering these exosomes to mice with allergic asthma, we want to find the function of FcεRIα-exosome on neutralizing IgE, reducing activation and quantity of MC, and also the immune regulation ability. Moreover, whether FcεRI and KIT signaling have relation with IgE level and receptor expression can confirm the function of FcεRIα-exo and find a new way for treatment of hypersensitivity and MC related diseases.

肥大细胞（MC）是过敏反应的效应细胞，IgE受体FcεR I和干细胞因子受体KIT是其膜标志，参与MC活化、分化和存活，两信号通路有协同效应。Exosome是靶分子和药物的理想载体。MCexosome（MC-exo）含胞内颗粒、mRNAs、携带FcεRI和KIT穿梭于细胞间隙，传递和释放炎症介质、细胞因子等发挥多种活性。FcεRIα链是IgE结合部位。基于对FcεRI和MC，MC-exo和DC等长期研究的基础，本课题拟从过敏性哮喘小鼠获取BMMC和FcεRIα转基因的imDC制备携带FcεRIα的exosome，输注给哮喘小鼠，通过分析、比较FcεRIα-exo作为FcεRIα载体中和游离IgE、减少MC活化和MC数量和exosome免疫调节的作用及其差异，FcεRI与KIT信号通路与IgE水平、受体表达的关系，明确FcεRIα-exo的作用机制，探索过敏甚至MC相关疾病治疗的新途径。

研究目的：分析肥大细胞（mast cell，MC）外泌体（MC-exo）的生物学特性，及其与游离IgE的相互作用，探讨MC-exo对MC活化和小鼠过敏性哮喘的抑制效果及机制。.研究方法：①SCF和IL-3诱导小鼠骨髓细胞分化为MC，差速离心提取MC-exo，电镜、免疫印迹（WB）、流式细胞及共聚焦显微镜鉴定MC-exo形态、蛋白表达。②质谱和核酸测序分析MC活化前后外泌体量、蛋白、核酸的差异。③将MC-exo和P815细胞系外泌体（P851-exo）与IgE共育，检测IgE与MC的结合、外泌体与IgE共育后对MC活化释放β己糖苷酶及组胺的影响以及活化信号蛋白表达。④建立BALB/c小鼠卵清蛋白(OVA)过敏性哮喘模型，检测尾静脉注射MC-exo 后1、2、3个月气道高反应(AHR)、血清OVA特异性IgE（OVA-sIgE)、肺泡灌洗液(BALF)炎症细胞、组胺、细胞因子变化，肺组织切片、HE和糖原染色分析病理改变。.研究结果：① SCF和IL-3体外诱导4周后小鼠骨髓细胞可分化为成熟MC并分泌外泌体。外泌体呈圆形囊泡状、50-100nm；PKH67染色证实外泌体可被MC摄取。② MC活化前后外泌体差异表达蛋白415个（共获得1988个）其中286上调、129下调，富集通路与溶酶体、神经系统病变、内质网蛋白加工相关。差异表达lncRNA61个（共获得397个）和miRNA47个（共272个）其中上调11个、下调36个。③ MC-exo能通过其负载的FcεRI与MC竞争结合游离IgE，并明显降低MC释放β己糖苷酶和组胺。MC活化信号通路中PLCγ1- PKC蛋白磷酸化减少。④MC-exo能有效减轻哮喘小鼠AHR，降低其血清和BALF的OVA-sIgE、组胺、2型细胞因子（IL-4、IL-5和IL-13）水平以及炎症细胞（嗜酸粒和淋巴细胞等）数量，而IL-10和IFN-γ水平增加。肺组织炎症细胞浸润、黏液分泌及气道平滑肌增生均减轻。.研究结论：①建立了体外培养和诱导BMMC、提取MC-exo方法；②MC活化前后外泌体含量、蛋白组学和转录组学均存在差异。③MC-exo负载的FcεRI通过结合游离IgE，下调PLC-PKC通路蛋白抑制MC活化。④MC-exo体内应用能有效缓解小鼠过敏哮喘症状、肺组织炎症和炎症因子。提示MC-exo具有过敏治疗的