基于树突状细胞递呈的牛乳过敏原致敏肽的研究

Dendritic cells (DCS) are specialized antigen-presenting cells, which are the key immune cells of food sensitization. In this program, the peptides from bovine allergens were digested by in vitro digestion, and the basic characteristics such as their molecular weight, amino acid sequence and composition were analyzed, which can be used to screen the potential allergenic peptide. Further, the Caco-2 cell model will be used to explore the effect of intestinal epithelial cells on digestive peptide transport by evaluating the peptide transmembrane operation rate, expression and distribution of intestinal connective protein and proinflammatory cytokines such as IL-8. In advance, the changes of dendritic cells uptaking and processing antigenic peptides will be detected by Morphological changes, antigen peptide uptake rate, surface markers and cytokines were evaluated, and induced by digestive peptides were characterized. Finally, the activation and difference of T lymphocytes will be monitored by flow cytometry and ELISPOT which is uses to detected the total stimulus molecule, signal transduction and transcription factor and so on.. By exploration of digested peptides from bovine protein digestion and peptide linkage of sensitization of dendritic cells and T lymphocytes, This project reveal the change regularity of bovine allergens related peptide digestive immune cells, so as to clarify the key role of dendritic cells in sensitization by milk protein and provide a better understanding of for mechanism of milk allergy.

树突状细胞是专职抗原递呈细胞，是食物致敏的关键免疫细胞。本项目拟通过体外模拟胃肠道消化乳过敏原，对消化肽的分子量和氨基酸序列等特性检测，筛选可能引起致敏的肽段；进一步通过Caco-2细胞模型，评估消化肽的跨膜运转率、肠细胞紧密连接蛋白的表达和分布以及促炎趋化因子IL-8等能力，探索肠上皮细胞对消化肽转运的影响；然后通过观察消化肽激发树突状细胞的形态变化、检测表面标志和释放的细胞因子以及抗原肽摄取率，阐明消化肽对树突状细胞的影响；在此基础上，采用流式细胞术和ELISPOT，监测T淋巴细胞的激活和分化所引起的共刺激分子、信号转导及转录因子等水平的变化。本项目通过探索乳过敏原蛋白消化肽对致敏过程中树突状细胞和T淋巴细胞的联动影响，揭示乳过敏原消化肽在致敏过程中引发相关免疫细胞的变化规律，从而阐明树突状细胞在乳蛋白致敏中的关键作用，为进一步深刻理解提供新的依据。

本项目对乳过敏原蛋白进行模拟体外胃肠道消化，检测消化产物的分子量及氨基酸序列，通过建立体外细胞模型和牛乳过敏动物模型来评价潜在致敏性肽段转运吸收能力，并分析树突状细胞在乳蛋白致敏中的作用。主要的研究结果如下：.1. 超高温灭菌乳、巴氏杀菌乳和脱脂乳三种乳制品经体外静态模拟胃肠道消化后。分别利用Tricine-SDS-PAGE和质谱对消化产物进行鉴定。结果表明，三种乳制品中的主要过敏原随着胃肠道消化时间的增加，逐渐降解。其中，酪蛋白在胃消化阶段被迅速降解。α-乳白蛋白和β-乳球蛋白具有较强的消化稳定性。液相-质谱技术分析10kDa以下的消化产物，进一步液相-质谱技术分析10kDa以下的消化产物，通过质谱对肽段的识别、分类，共检测到730条消化肽段。β-乳球蛋白的消化肽段集中的区域是AA1-21，AA23-61，AA71-103，AA107-119，AA123-140和AA148-160。.2. 体外利用牛乳蛋白致敏大鼠肠道对消化产物进行转运吸收，利用液相-质谱分析出 9 条能够进入肠道黏膜系统并具有潜在致敏性的肽段，包括 β-乳球蛋白中 AA61-69，AA125-135，AA125-138，AA92-101及AA149-162；αS1-酪 蛋 白 中 AA84-90和AA125-132；αS2-酪蛋白中 AA25-32。.3. 利用Caco-2细胞单层模型评估牛乳过敏原消化肽段的转运吸收能力。结果表明，与消化产物相比，合成β-乳球蛋白来源的多肽（AA1-14，AA24-35，AA40-60，AA82-101，AA123-139）具有良好的转运吸收能力。.4. 分离获得β-乳球蛋白致敏的小鼠地固有层和肠系膜淋巴结，利用流式细胞术检测消化肽段与细胞共孵育后细胞表型的变化。结果表明，β-乳球蛋白来源的肽段AA40-60能显著诱导树突状细胞成熟。肽段AA1-14，AA24-35和AA40-60诱导T细胞向Th1方向分化，肽段AA82-101和AA123-139诱导T细胞向Th2方向分化。