特异性T细胞表位含量对新疆小麦麸质蛋白乳糜泻致敏性的影响

Populations from Xinjiang is at high risk of celiac disease. Since abundant genetic diversity of storage proteins in wheat landraces exists in this area, wheat varieties with low allergenicity related to celiac disease should be discovered. In this proposal, the effect of different level of specific gluten T-cell epitopes on allergenicity related to celiac disease of wheat varieties from Xinjiang will be investigated. Firstly, the level of celiac disease-related gluten T-cell epitopes will be determined, and wheat varieties will be typed for their level of T-cell epitopes. Then, three type of wheat varieties with high, middle and low level of T-cell epitopes will be used for gluten extracting, respectively. The digestibility of gluten will be estimated by in vitro gastrointestinal digestion, and it was used to test the absorption and transport of digestion products. The allergenicity related to celiac disease of digestion products will be estimated by Caco-2 cell inflammatory response and T cell proliferation assay. Afterwards，a rat model for celiac disease will be developed. Celiac disease-related cytokines, NFκB mRNA and COX-2 mRNA expression, intestinal histopathology will be estimated to assess the allergenicity of the three type of wheat varieties with different content of T-cell epitope related to celiac disease..Through the project, the relationships between the level of specific gluten T-cell epitopes and allergenicity related to celiac disease of gluten will be clearly elucidated. It will be a strong academic support to evaluate allergenicity related to celiac disease of wheat varieties based on the level of specific gluten T-cell epitopes. Moreover, it will be successful in digging hard for wheat varieties with low allergenicity related to celiac disease, providing a safety material for processing gluten-free food.

新疆地区是乳糜泻发生的高风险地域，而且小麦种质遗传多样性丰富，可能存在低乳糜泻致敏性的种质资源。以新疆小麦麸质蛋白为研究对象，首先采用免疫印迹法和RNA扩增子测序法定量麸质蛋白中致乳糜泻T细胞表位含量；其后，选取高、中、低T细胞表位含量的3个小麦品种，提取麸质蛋白，体外模拟消化测定麸质蛋白耐消化能力，并研究消化产物在Caco-2细胞模型的吸收转运，再用Caco-2细胞炎性反应实验和T细胞增殖实验评价消化产物的乳糜泻致敏性；然后，构建乳糜泻大鼠模型，通过检测炎性细胞因子、NFκB mRNA和COX-2 mRNA表达量、肠道组织病理变化，评价3种不同T细胞表位含量的麸质蛋白的乳糜泻致敏性。.通过本项目研究，可以阐明T细胞表位含量与麸质蛋白乳糜泻致敏性的关系，为基于麸质蛋白T细胞表位含量评价乳糜泻致敏性提供可靠的科学依据；同时期望发掘低乳糜泻致敏性的小麦种质资源，进而为加工无麸质食品提供特需原料

本项目以新疆小麦麸质蛋白为研究对象，采用免疫印迹法和RNA扩增子测序法定量麸质蛋白中致乳糜泻T细胞表位含量；然后结合麸质蛋白消化稳定性和基于细胞和动物实验的乳糜泻致敏性评估，阐明T细胞表位含量与麸质蛋白乳糜泻致敏性的关系。研究的主要方法、结果及结论如下: .1．采用免疫印迹法检测164种新疆小麦麸质蛋白乳糜泻致敏性 T 细胞表位含量，选定高、中、低乳糜泻致敏性T细胞表位含量小麦品种各3种。再采用RNA 扩增子深度测序法、及体外消化实验结合质谱法检测9种小麦品种中乳糜泻致敏性 T 细胞表位含量。三种方法的结果存在一定差异性。.2．选取上述三种检测方法均显示乳糜泻表位含量存在较大差异的两个小麦品种01-25和04-引鉴105，进行Caco-2 细胞实验和动物实验。.3．Caco-2 单层细胞跨膜转运实验及炎性实验结果显示：与04-引鉴105相比，01-25可降低紧密连接蛋白Occludin的表达，增加通透性；与01-25比较，04-引鉴105可明显抑制炎性通路蛋白NFκB P65和P38磷酸化的表达，而01-25可明显增加NFκB P65、P38、P44/42和P-P44/42蛋白的表达，升高IL-6水平。.4．麸质蛋白乳糜泻致敏性的动物实验结果显示：麦醇溶蛋白可明显减轻免疫器官胸腺脏器指数，显著增加特异性抗体IgG、IgA水平，调节血清、脾细胞、空肠裂解液中乳糜泻相关细胞因子IFN-γ、IL-10、IL-15、IL-17A、IL-21、IL-4、IL-6和TNF-α的水平，引起十二指肠、空肠、回肠绒毛扁平化，部分萎缩，隐窝数量减少。01-25与04-引鉴105比较，01-25对上述指标的影响更明显。.5．研究结果显示：体外消化结合质谱法的T细胞表位含量检测结果与细胞和动物实验结果更吻合，更适用于体外评估麸质蛋白乳糜泻致敏性。