超高压处理降低花生Ara h 2致敏性的分子机制研究

Peanut is an important food allergen, which has been paid more and more attention because of the potential risk, long term and increasing incidence. Ara h 2 is the most sensitive protein in peanut allergen and it is significant to study and reduce the sensitivity of Ara h 2. High hydrostatic pressure (HHP) treatment can effectively reduce the sensitivity of Ara h 2, but the mechanism is not clear. In the research, the peanut allergy mice model was established, based on RNA-sep to construct transcriptome sequencing database of Ara h 2 in intestinal of allergic mice before and after HHP treatment. Illumina high-throughput sequencing was used to analysis of gene annotation, functional classification of GO and COG through bioinformatics technology and clarify KEGG metabolism way and systematicly analyzed the key gene involved in type I allergic reaction pathway. Associated candidate gene and molecular marker were obtained of Ara h 2 treated with HHP, Ara h 2 mediated signal pathway related to allergic reactions was also analyzed. In addition, the expression of genes associated with differences were found in Ara h 2 before and after high pressure treatment the mice, which reveals the molecular mechanism of reducing allergic reaction of Ara h 2 treated with HHP.

花生是重要的食物过敏原，花生过敏反应因其潜在的危险性、长期性以及不断增加的发病率而日益受到重视。Ara h 2是花生过敏原中致敏性最强的蛋白，研究并降低Ara h 2致敏性具有重要意义。超高压处理能有效降低Ara h 2的致敏性，但作用机制尚不清楚。本课题以建立的花生过敏小鼠模型为研究对象，基于RNA-sep的转录组学技术，构建超高压处理前后Ara h 2过敏小鼠肠道的转录组测序文库，利用Illumina高通量测序，通过生物信息挖掘分析获得相应的基因注释、GO和COG功能分类，明确KEGG代谢途径，系统分析参与Ⅰ型过敏反应信号途径的关键基因序列，获得超高压处理后Ara h 2致敏性降低相关联的候选基因与分子标记，解析Ara h 2介导过敏反应的相关信号通路，证明关联基因在超高压处理Ara h 2前后小鼠机体的差异表达，进而揭示超高压处理降低花生Ara h 2致敏性的分子机制。

本项目首先构建花生过敏小鼠模型，通过口服HHP处理后花生主要过敏原蛋白Ara h 2发现，与过敏组相比，小鼠过敏临床评分显著降低，血清特异性IgE及组胺含量均显著降低，且与对照组差异不显著，因此过敏小鼠在灌胃超高压处理后的Ara h 2，其过敏症状明显缓解。该研究结果显示，HHP处理后Ara h 2对花生过敏小鼠致敏性显著降低，提示超高压协同温度处理可作为一种有效控制花生Ara h 2致敏的处理方式。其次基于RNA-sep的转录组学高通量测序技术，成功筛选到HHP处理后与过敏组差异的446个基因，其中上调差异基因300个，下调差异基因146个，进行GO功能分类以及KEGG代谢途径分析过敏反应信号途径与Toll样受体（TLRs）、核因子（NF-κB）、T细胞、B细胞等密切相关。最后，采用RT-PCR技术验证关键信号分子基因表达，结果表明，超高压处理组小鼠TLR2、TLR4、NF-κB、IL-4、IFN-γ和IL-10 mRNA相对表达量均显著低于过敏组，与对照组差异不显著，说明超高压处理降低Ara h 2致敏性通过TLR2/TLR4-NF-κB信号通路，下调炎性分子表达，恢复Th1/Th2平衡，从而启动T、B效应细胞控制过敏的发生。