鸡蛋卵运铁蛋白的胃肠消化及其在致敏中的作用机制

Food allergy is usually induced mainly through the gastrointestinal route, and gastrointestinal digestion plays an important role in the development of food allergy. In this proposal, egg ovotransferrin (OVT), one of the main egg allergens, will be investigated in an in vitro static digestion model for infants/adults. The digestibility of the egg OVT, the structure and IgE binding ability of the digested OVT will be tested, and the relevance to allergenic epitopes of the digestion products will be also analyzed. Afterwards, Caco-2 cell model will be used to detect the rate of transepithelial transport of the digested OVT, as well as the amount of tight junction protein and interleukin 8 in the stimulated cell, and all the tests will provide an assessment to the influence of the digestion products on intestinal epithelial barrier. The peripheral lymphocyte proliferation, basophils histamine release, and the assessment of the antigen processing capacity by dendritic cell will be carried out to define the allergenicity of the OVT digestion products. Furthermore, a Balb/c mice model will be used to evaluate the allergenicity of the digested OVT by testing the specific IgE, lymphocyte proliferation and relevant cytokines related to the food allergy. .This work aims to illustrate well the influence of simulated gastric/intestinal fluid on the digestion of OVT and the role of digestion products in the key point of allergic reaction. Accordingly, the allergic mechanism of the digested OVT will be stated clearly, providing fundamental knowledge for deeply understanding the role of gastrointestinal digestion in the development of OVT allergy.

食物过敏原主要通过肠道致敏，胃肠消化对其致敏性具有重要作用。本项目以鸡蛋卵运铁蛋白为研究对象，模拟婴幼儿/成年人胃肠液并用静态消化模式评价卵运铁蛋白的消化稳定性，解析消化产物结构并揭示它与致敏性表位的关联性，检测消化产物的特异性IgE结合力；采用Caco-2细胞模型，测定跨膜转运率、肠细胞紧密连接蛋白及促炎趋化因子IL-8，探索消化产物对肠上皮细胞屏障的影响；以树突状细胞为模型，测定树突状细胞对消化产物的摄取率和消化产物的组织蛋白酶降解特性，评估抗原加工性能；经嗜碱性粒细胞的组胺释放实验，评价消化产物激活致敏细胞的能力；采用小鼠致敏模型，检测一系列与食物过敏相关的抗体和细胞因子，评价消化产物的致敏性。.本项目将探明模拟胃肠液对鸡蛋卵运铁蛋白的消化影响及消化产物在过敏反应关键环节中的免疫学功能，诠释消化产物在卵运铁蛋白过敏中的工作机制，为深刻理解胃肠消化在卵运铁蛋白过敏中的作用提供理论依据。

在体外静态消化模式下，分别模拟婴幼儿和成年人的胃液、小肠液以及肠刷状缘酶消化鸡蛋卵运铁蛋白(OVT)，并利用Tricine-SDS-PAGE、MALDI-TOF-MS和RP-HPLC对消化产物分别进行分析，评估其消化稳定性，结果发现，成年人模拟消化OVT的能力比婴幼儿强，BBM酶在食物过敏原消化过程中具有重要作用。通过体外血清学ELISA实验测定卵运铁蛋白及其消化产物与特异性IgE、IgG的结合能力，评估了其潜在致敏性，并采用Western-Blotting的方法筛查OVT消化产物与IgG的结合片段，基于体外血清学初步阐明了OVT消化产物与特异性抗体的结合作用。采用Caco-2细胞单层膜模型，测定了其跨膜转运能力，探明了OVT及其体外胃肠消化产物对细胞屏障的影响。进一步通过OVT及其消化产物对嗜碱性粒细胞的相关细胞因子释放能力、对外周血单核细胞增殖的影响，以及树突状细胞对它们的摄取，从体外细胞学方面综合评价了致敏性,为解释OVT消化产物的致敏能力提供了科学数据。最后，以Balb/c小鼠为研究对象，模拟食物过敏发生过程中致敏与激发两个关键阶段，对实验动物的临床表现进行评分，检测分析了小鼠的特异性抗体含量，观察了肺部及肠道组织变化，评价了OVT及其消化产物的致敏性。通过本项目的研究，探索了OVT消化产物在过敏反应各阶段中的免疫功能变化，揭示了OVT致敏的动态过程，更加科学地认识胃肠消化在食物过敏中的重要作用。