基于树突状细胞成熟及其表观遗传调控机制的中医过敏体质研究

Allergic diseases have a high incidence and they do much harm to human health. The common fundamental pathomechanism of allergic diseases is allergic reaction. Recent research show that the maturation of dendritic cells (DCs) play a important role in the activation of allergic reaction. As a bridge between innate immunity and adaptive immunity, dendritic cells have the function of inducing allergic reaction and maintaining immune balance. Allergic constitution of TCM is the status before allergic diseases. On the basis of former study, we propose the following hypothesis: The mechanism of allergic constitution exits in the defective maturation of dendritc cells and that result in the wrong function of DCs. And then lead to the result that human body is allergic to allergens in the outside world. We call that "immune sub-steady". Under the guidance of such hypothesis, in this study, we decide to research the function and maturation of DCs in people with allergic constitution. The classic model of lipopolysaccharide (LPS )-inducing DC maturation will be used in this study. The difference of gene expression between immature DC and mature DC will be detected with the technique of immunity chip array. And the epigenetic regulating mechanism of DC will be detected with the technique of DNA promoter methylation sequencing. Some people with allergic constitution cured by oral Chinese herbal medicine will be designed to verify the regulating mechanism of DNA promoter methylation. Finally, we will summarize the above research results, to expound the action of DC, its maturation and regulation in allergic constitution, so as to provide the experimental evidence for diagnosis and assessment of allergic constitution, and give certain guidance to prevent and treat allergic diseases in earlier stage from the perspective of TCM constitution.

以变态反应为病理基础的过敏性疾病是危害人类健康的多发病。树突状细胞（DC）由于其成熟前后功能演变的特殊性，作为固有免疫和特异免疫的桥梁，在诱导变态反应和维持免疫平衡中起主要作用。过敏体质是过敏性疾病的"未病"状态，基于前期研究结果，我们提出"过敏体质是由于DC成熟和调控机制失常导致其功能缺陷，使机体易被过敏原致敏，处于免疫'亚稳态'"的假说。本研究拟以DC功能为切入点，以DC成熟和调控机制为研究主线，以过敏体质的"未病"层面为理论指导，设立平和体质对照组，采用体外LPS诱导DC成熟的经典模型，以免疫芯片技术分析两组外周血DC成熟前后基因表达的差异；以DNA甲基化测序技术分析DC成熟的表观遗传调控机制，并设中药干预后脱离过敏体质者进行反证。整合以上点、线、面研究结果，明确DC功能、成熟和调控机制在过敏体质形成中的作用，为临床过敏体质判定提供实验依据，并为从体质角度早期防治过敏性疾病提供指导。

以变态反应为病理基础的过敏性疾病是危害人类健康的多发病。过敏体质是过敏性疾病的“未病”状态。本项目提出“过敏体质是由于DC 成熟和调控机制失常导致其功能缺陷，使机体易被过敏原致敏，处于免疫‘亚稳态’”的假说。以DC 功能为切入点，采用体外诱导DC 成熟的模型，观察过敏体质与平和体质DC成熟前后形态和功能的差异，并以免疫芯片技术分析两组外周血DC 成熟前后基因表达的差异。①应用GM-CSF、IL-4和TNF-α联合诱导培养树突状细胞。观察细胞成熟前后形态，进行混合淋巴细胞反应及细胞表面标志检测，研究过敏体质DCs形态及功能特点。结果：过敏质成熟DC数目，淋巴细胞增殖能力及成熟DC的CD80、CD86表达，未成熟DC的CD14、 HLA-DR表达显著高于平和质，提示过敏质DC功能的异常。过敏康干预后过敏体质组DC功能部分恢复。②成熟前后的DCs差异基因表达分析，对差异基因进行GO和Pathway富集，阐释其部分差异表达基因通路与DCs成熟的关系，探讨过敏体质DCs成熟前后功能及非编码RNA特征。结果：mRNA芯片检测显示过敏质和平和质DC成熟前后基因表达有显著差异，GO富集显示过敏质和平和质未成熟DCs有信号转导、膜组成、蛋白质同源二聚化活性的功能差异，成熟DCs有免疫应答、膜组成及腺苷三磷酸结合的功能差异。pathway富集显示过敏质和平和质未成熟DCs有吞噬体通路的异常，成熟DCs有细胞粘附分子通路的异常。说明过敏体质未成熟DCs有较强的吞噬能力，成熟DCs细胞粘附能力较强，参与免疫应答，抗原提呈能力较强。经RT-qPCR验证，成熟树突状细胞平和质对比过敏体质mRNA表达HLA-B呈上调趋势、PTAFR呈下调趋势。未成熟树突状细胞平和质对比过敏体质HLA-C、MARCO、KIR2DS3呈上调趋势、IFNB1呈下调趋势。结论：相对于平和体质者，过敏体质者的树突状细胞存在功能的异常，基因表达谱存在显著差异。本项目从树突状细胞功能、成熟及调控机制的角度研究过敏体质人群的免疫状态，揭示过敏体质形成的分子机制，为临床过敏体质判定提供实验依据，并为从体质角度早期防治过敏性疾病提供指导。