鱼类小清蛋白致敏性与耦合细胞体系交互作用关系的研究

In recent years, Food allergy has become a food safety issue of international concern. There is an urgent need to conduct a risk assessment to develop practical safety standards and measures for overcoming the food allergy concern. But the mechanism of allergic reaction is complex; an evaluation which focused on one factor alone cannot fully reflect the role of allergen in the sensitization process. It is difficult to meet the current allergenicity evaluation requirements of food allergens. Recent studies show that the interactions between the desensitization of dendritic cells and the activation of mast cells are essential to characterize the allergenicity. In this study, the major fish allergen-parvalbumin will be taken as the research object to establish a coupled co-culture system of dendritic cells and mast cells. The effects of parvalbumin on metabolism of the coupled cell system from antigen-presenting expression, signal transduction, transcriptional translation and final response will be analyzed; further research focuses on analyzing the dynamic changes of typical metabolites, and to explore its quantitative relationship with the parvalbumin. In this way, the relationship between the interaction of coupled cell system and the allergenicity will be clarified. Finally, combining with mice allergy model, the evaluation model of fish parvalbumin allergenicity based on coupled cell system will be established. This model could provide theoretical and methodological bases for establishing risk assessment system of food allergens and controlling the risk of food allergy worldwide.

近年来食品过敏已成为国内外重点关注的食品安全问题，迫切需要对食品过敏原进行风险评估以制定切实可行的安全标准，但过敏反应机制复杂，单独针对某一环节的评价方式不能充分体现过敏原在致敏过程中的作用，难以满足当前对过敏原致敏性评价的要求。最新研究表明，机体中致敏环节的树突状细胞与激发环节的肥大细胞交互作用在诱导过敏反应中至关重要，是表征食品过敏原致敏性的关键。本课题拟以我国主要的过敏原——鱼类小清蛋白为研究对象，构建树突状细胞和肥大细胞的耦合共培养体系，研究小清蛋白对耦合细胞体系从抗原递呈、信号传导、转录翻译到最终响应等各阶段代谢的影响，解析典型代谢产物的动态变化规律，探讨其与小清蛋白的定量关联机制，阐明树突状细胞和肥大细胞交互作用与致敏性的关系；进一步结合小鼠过敏模型，创建基于耦合细胞体系的鱼类小清蛋白致敏性评价模型，为我国建立食品过敏原的风险评估体系，控制食品过敏风险提供理论依据和方法学基础。

食品过敏是国内外重点关注的食品安全问题，但如何全面准确的对食物过敏原的致敏性进行评价一直是业界的难题，使得无法对食物过敏原的风险进行充分评估，严重影响了食品工业中过敏风险的消除，因此迫切需要建立更加完善的食物过敏原致敏性评价方法。因此本项目以我国主要的过敏原——鱼类小清蛋白为研究对象，通过构建树突状细胞和肥大细胞的耦合共培养体系，系统研究了鱼类小清蛋白对耦合细胞体系从抗原递呈、信号传导、转录翻译到最终响应等各阶段代谢的影响，发现小鼠骨髓源树突细胞（BMDC）模型具有作为过敏原潜在致敏性评价模型的潜力，该模型的潜在生物标志物为表型分子MHC-Ⅱ、CD86以及细胞因子IL-6、IL-12p70、IL-23、IFN-γ；为了更全面的评价致敏性，建立了BMDC与CD4+ T细胞耦合共培养模型，确定了以IL-4、IFN-γ等细胞因子水平为生物标志物，继而实现对T细胞的Th1或Th2偏移方向鉴别。通过对评价模型进行优化，发小清清蛋白的浓度范围为0 - 0.5 mg/mL，BMDC细胞浓度为2×106个/mL，抗原激发时间为48 h，BMDC与CD4+ T细胞比例1:10为最适条件，此时模型体系中各细胞因子水平最稳定。进而分析稳定细胞模型中这些典型代谢物的动态变化规律，建立了典型代谢产物与致敏性指标的定量关系，并形成了树突细胞模型的标准操作程序和耦合细胞致敏性评价模型的标准操作程序。在此基础上，利用传统评价手段（血清学评价、体外抗胃肠消化研究、小鼠过敏模型评价、RBL-2H3细胞模型、KU812细胞模型）以及新型细胞模型（BMDC细胞评价模型和以BMDC与T细胞为基础的耦合共培养细胞评价模型）分析糖基化对大菱鲆PV致敏性的影响。通过综合分析各评价模型的结果，发现建立的新型细胞评价模型能够较好的与传统评价模型结果相一致，验证了基于致敏阶段细胞的免疫学新型评价模型评价结果的正确性和可行性。以上研究结果为我国建立食品过敏原的风险评估体系，控制食品过敏风险提供理论依据和方法学基础。