表观遗传学修饰Treg细胞经区域淋巴结诱导鼻粘膜免疫耐受的研究

The sharp increase in allergic disease prevalence over the past 2 or 3 decades is difficult to explain by genetics,so epigenetics as a mechanism mediating gene-environment interaction got great attention. Regional lymph node acting as the hub of the local immune response should be highly valued.This project is based upon the key role of Tregs in inducing allergic rhinitis (AR) immune tolerance,plans on utilizing Tregs modified by epigenetics to inducing immune tolerance of allergic nasal mucosa via regional lymph node in order to find new method of treatment of AR. Concrete research content include: ①analyze the epigenetic modification of Tregs in AR by using Massarry, RT - PCR and the downstream immune response regulation.②detect the correlation between some environmental factors ,such as PM2.5、PM10,and the methylation/demethylation of foxp3 which is the key factor of Tregs.③induce foxp3 demethylation of allergen specific Tregs in vitro by using demethyl-agent.④allergic animal models will be performed to test whether epigenetics modified Tregs could induce immune tolerance of allergic nasal mucosa after regional lymph node injection and then detect epigenetics phenomenon of its daughter. The object is explaining the pathogenesis of AR from epigenetics perspective and its correlation with environmental pollution,thereby we will address future challenges and opportunities for the new field,focusing on those that might have potential clinical therapeutic role in preventing allergic disease.

目前，单以遗传学说难以解释不断升高的变应性疾病发病率而使表观遗传调控受到极大关注。区域淋巴结作为调控局部免疫应答的枢纽未引起足够重视，本研究基于Tregs在诱导变应性鼻炎（AR）免疫耐受中的关键作用拟通过表观遗传修饰Tregs经区域淋巴结诱导变应性鼻粘膜的免疫耐受，以期寻找治疗AR的新方法。本课题研究内容：①通过Massarry、RT-PCR等方法检测AR中Tregs表观遗传修饰及对下游免疫应答的调控；②检测PM2.5、PM10等环境因素对Tregs关键因子foxp3甲基化/去甲基化的影响；③利用去甲基化剂体外诱导变应原特异性Treg细胞foxp3去甲基化；④通过动物模型探讨表观遗传修饰后的Tregs经区域淋巴结诱导免疫耐受并观察其子代的表观遗传现象。以期从表观遗传学角度解释AR的发病及与环境污染的关系，从而探讨表观遗传调控Tregs诱导免疫耐受在AR治疗中的作用和潜在临床可行性。

AR是引起哮喘发病的重要因素，而全球有近5亿、我国近1.5亿人罹患AR, 因此近年对该病的发病机制、诊断及治疗均进行了大量研究以找出遏制流行率上升趋势的有效预防策略；迄今为止，研究结果虽然取得瞩目成果，却仍未能控制发病率的快速增加，而治疗方法（抗介质、抗炎和特异性免疫）数十年来也无大的突破。鼻用糖皮质激素和抗组胺等药物治疗，较好的控制了AR症状，但不能改变自然病程；标准化变应原免疫治疗提高了AR的治疗水平，但疗程长，依从性差，且安全性存在争议。虽然这些治疗在控制大多数病人症状上有效,仍有20%-40%的患者未能很好的控制症状,而且很多患者需终生用药，另外部分患者担心激素治疗的副作用。另外，AR的治疗也已成为预防或改善过敏性哮喘的必要手段，因此控制变应性鼻炎发病率的持续增加、寻找新的治疗策略仍是我们面对的巨大挑战。探索新的可能的治疗途径和靶点,也是本课题的研究意义。为了探讨鼻黏膜上皮细胞与Treg细胞之间是否直接存在直接的信息传递，建立鼻黏膜上皮细胞- CD4+CD25+Foxp3+Treg细胞体外共培养模型。采集健康志愿者鼻黏膜上皮细胞，进行人类原代鼻黏膜上皮细胞体外培养至细胞融合，免疫磁珠分离健康志愿者人外周血单个核细胞（peripheral blood mononuclear cell, PBMC）来源的CD4+CD25+Foxp3+ Treg细胞，建立NECs-Tregs共培养模型。流式细胞术检测共培养后与单独培养的Treg细胞的细胞表面标志的表达，探讨与单独培养的Tregs相比较共培养后Treg细胞抑制性活性的细胞表面标志CCR8、CTLA-4表达增高（p=0.002,P＜0.05;p=0.035,P＜0.05）的意义。第二部分：筛选并探讨AR鼻黏膜上皮细胞暴露于特异性变应原后可能与Treg相关的细胞因子的表达水平的变化及意义。采集血清特异性尘螨IgE阳性的变应性鼻炎患者及健康志愿者鼻黏膜上皮细胞体外培养。RT-PCR检测经特异性变应原重组蛋白片段Der p1刺激后AR组与对照组可能与Treg相关的细胞因子的mRNA的表达水平，主要包括TSLP，IL-10，TGF-β， IL-25， CCL1；ELISA检测细胞悬液中的蛋白浓度。分别探讨AR组经Derp1刺激后与健康对照组及空白对照组相比较TSLP 表达增高（P＜0.05）、CCL1表达降低（P＜0.05）