肥大细胞源exosomes特性分析及其负载OVA对过敏性哮喘的免疫调节作用

Mast cell derived exosomes(MCEX), containing cytoplasm inclusion within the MC and expresses MC membrane molecules, is a carrier that the MC uses to transmit information and cell extracellular matrix with other cells, and transfer molecules through miRNAS regulation the proteins experesion in other cells. .MCEX and allergen loaded MCEX have been successfully abstracted in our preliminary experiments..This project tries to explore 1. To define the components of MCEX and their biological activities by extracting exosomes from mouse BMMC and by analyzing their miRNAs, enzymes, inflammatory mediators, CKs and membrane proteins;.2. To explore the mechanism of MCEX regulating Th,through comparison between MCEX co-cultured with CD4 + T and medium only cultured CD4 + T,analyze the regulation of MCEX to Th's differentiation and proliferation, the capacity of producing CKs; compare the differences in the miRNAs of the exosomes drived from Th cells..3. MCEX load OVA adaptive injecte to normal and asthma model mice, then observation of asthma symptoms of mice, analysis of the level of CKs, inflammatory mediators, IgEand Th1/T2 ratio in BALF and serum of the mice, expression of the FcεRI and expersion of the PBMC..The subject explore MCEX load antigen-induced allergen tolerance and reduce of allergic asthma and its mechanisms. .In the study we would recognize the role of MCEX in allergic reactions deeply, and seek the key regulation of miRNA and explore the possibility of MCEX as an allergic vaccine or treatment for allergic reactions.

MCEX含MC胞内物、表达MC膜分子，是MC与其它细胞和胞外基质传递信息、转移分子、通过miRNAS调控其它细胞的载体。预实验已得到MCEX和过敏原负载的MCEX。本课题拟1.从鼠BMMC提取exosomes，分析其中主要的miRNAs、酶、炎症介质、CKs和膜蛋白，确定MCEX的成分，判断其生物活性；2. 将MCEX与CD4+T共育与单纯培养的CD4+T比较MCEX对Th分化、增殖、产生CKs能力的调节；比较Th细胞exosomes的miRNAs差异，探讨作用机制；3. MCEX负载OVA回输至正常和哮喘模型鼠，观察哮喘症状，分析BALF和血清CKs、炎症介质、IgE、Th1/T2比例，PBMC FcεRI表达等。探讨MCEX负载抗原诱导过敏原耐受和抑制过敏性哮喘的作用及其机制。深入认识MCEX在过敏反应中的作用，寻找调控的关键miRN并探讨MCEX作为过敏原疫苗或治疗过敏反应的可能性。

研究背景：肥大细胞（MC）是过敏反应的效应细胞，又有多种免疫活性。MCEX含MC胞内物、表达MC膜分子，是MC与其它细胞和胞外基质传递信息、转移分子、通过miRNAS调控其它细胞的载体。.研究内容：从小鼠肥大细胞中提取exosomes（MCEX），分析其中主要的核酸和蛋白，确定MCEX的成分，判断其生物活性；评估MCEX对Th分化、增殖、和功能的影响；探讨MCEX在肿瘤中的作用及机制。.研究结果：小鼠骨髓细胞在SCF的诱导下经4周培养可以分化为肥大细胞。通过超高速离心法制备了MCEX。MCEX表达exosome特征性分子（CD81、TSG101）和MC特征性分子（FcεRI、KIT、OX40L），含有丰富的microRNA。MCEX能以其表面的OX40L与T细胞表面的OX40结合，进而促进naive CD4+T细胞的增殖和向Th2方向分化。此外，我们的研究还表明，MCEX将KIT蛋白转移至非小细胞肺癌，并通过活化非小细胞肺癌细胞内PI3K/Akt信号通路上调细胞周期蛋白表达，促进肿瘤细胞的增殖及加速肿瘤细胞周期等生物学行为。.科学意义：获得了MCEX的成分数据，初步明确了MCEX对T细胞、肿瘤细胞的作用。既为MXEC负载OVA治疗过敏的研究奠定了基础，也为MC及其exosome在肿瘤的作用及机制研究提供了依据。