Der p1/IgG融合蛋白对树突状细胞表型和功能调节及机制研究

Allergen vaccacine with genetic-engineered modification is the hot topic for immunotherapy of allergic disease. DC expresses two types of specific immunoglobulin receptors simultaneously: FcεRI for IgE and FcγRIIb for IgG respectively. The FcεRI-antigen-FcγRIIb cross-linking can lead immunosuppressive signal in DC and subsequent inhibition of allergic response. In this study, we plan to construct a fusion protein containing Derp 1 and IgG Fc fragment, namely Derp1/IgG fusion protein, to investigate whether this modified fusion protein have ability to induce tolerogenic DC phenotypy and inhibition of allergic inflammation.Moreover, considering miRNAs play an important role in the regulation of innate and adaptive immune responses,and we have previously identified several miRNAs involved in the regulation of maturation and function of DC, we herein will analyze the change of miRNA profiles in DC in response to Derp1/IgG protein, and identify the key miRNAs that participated in the post-transcriptional regulation of FcεRI and FcγRIIb receptor and subsequently cross-link activation signal molecule.In dosing so, this study is expected to clarify the underlying mechanism of FcεRI and FcγRIIb receptor and subsequent cross-link activation signal molecule to regulate T cell differentiation by Derp1/IgG fusion protein and to comfirm the effect on allergic rhinitis of therapy by Derp1/IgG fusion protein, which will be beneficial for the application of the Derp1/IgG fusion proteinc in treating allergic diseases.

采用基因工程技术修饰疫苗是变应性疾病免疫治疗研究的热点问题。根据树突状细胞（DC）表面同时表达FcεRI（IgE受体）和FcγRIIb（IgG受体），以及FcεRI-抗原-FcγRIIb交联能引发DC的免疫抑制性信号的研究报道，本课题拟采用IgG片段修饰变应原Derp1构建Derp1/IgG融合蛋白，观察能否诱导DC形成耐受型表型从而抑制变应性炎症。同时，鉴于miRNA在免疫反应的调控中发挥重要作用且本课题组已证实多个miRNA参与并调控变应原诱导的DC成熟及功能。本课题拟分析Derp1/IgG诱导耐受型DC过程中miRNA的表达谱系，确定参与FcεRI和FcγRIIb受体及其交联激活后引发信号的转录后调控的关键miRNA，阐明FcεRI和FcγRIIb交联激活后对T细胞分化的影响和机理及其转录后调控机制，验证Derp1/IgG在变应性鼻炎治疗中的作用，用于未来变应性疾病的治疗。

变应性鼻炎是一种Th2细胞优势分化的黏膜慢性炎性病变，本课题检测了中国人变应性鼻炎患者的临床表型特征，评估了变应性鼻炎患者外周血差异表达的miRNA；在此基础上，采用IgG 片段修饰变应原Derp1 构建Derp1/IgG 融合蛋白，检测了融合蛋白对树突状细胞表型的作用、Treg细胞的诱导作用及对变应性气道炎症的抑制作用。并以miR-106b、miR-146a、miR-17等为研究靶点，探索miRNA在变应性鼻炎发病及免疫治疗中的作用；.研究发现，证实中国南方的变应性鼻炎患者多数对一种以上的过敏原过敏，尘螨是其中最主要的变应原；研究发现共刺激分子TIM-1 mRNA在变应性鼻炎患者表达增高，经过半年尘螨舌下免疫治疗后显著降低，TIM-1 mRNA表达降低的情况与血浆IL-5、ECP浓度的降低直接相关；发现miR-146a在变应性鼻炎患者外周血PBMC显著降低，经过3个月舌下和皮下尘螨免疫治疗后显著增高，Treg 细胞数量增加，证实miR-146a可以作为变应性鼻炎免疫治疗的重要参考指标；通过检测了离体培养的树突状细胞在变应原OVA的刺激下miR-106b显著下调，证实miR-106b能够通过作用于EGR-2参与Th2反应的抑制过程，提示EGR-2可能是miR-106b调控变态反应发生的靶点；采用IgG 片段修饰变应原Derp1 成功构建了Derp1/IgG 融合蛋白，并予以表达、纯化；通过融合蛋白刺激离体培养的树突状细胞，发现了一系列上调和下调的miR。发现融合蛋白刺激后的树突状细胞能够诱导miR-146a依赖的Foxp3+ Treg细胞产生，而转染miR-146a的树突状细胞对气道炎症具有抑制作用；证实息肉组织中GSK-3β表达与Foxp3存在负相关，证实GSK-3β能通过磷酸化Foxp3调节Treg细胞的稳定性；证实了炎症条件下由IL-6/miR-17信号通路介导的一种非Foxp3依赖性Treg细胞免疫活性的调控机制，即miR-17通过负调控Eos在内的Foxp3相关共调节分子的表达而抑制Treg细胞抑制功能的发挥。整个研究从基因工程疫苗通过miRNA调节树突状细胞的层面上进一步拓展了学术界对变应性鼻炎发病机制，对提升免疫治疗的临床疗效具有重要的意义。