miR-395通过TSLP调节变应性鼻炎Th2反应

Numerous studies have shown that allergic rhinitis is a disease characterized by Th2 cell differentiation and subsequent eosnophilia. MicroRNAs are known to be involved in Th2 differention, and TSLP has been proven to be regulator of Th2 differentiation, while TSLP has been regarded as a target of microRNA-395 by bioinformatics technology. Therefore, we hypothesize that microRNA-395 is able to regulate Th2 differention by targeting TSLP in allergic rhinitis. Our study plan to focus on the difference of microRNA expressions in patients' nasal tissue by testing microRNA profiles, eosnophil infiltration，Th2 cytokines and TSLP levels, and analyze their correlations, next we plan to identify the immunological relationship of these factors in nasal tissue by focusing on microRNA-395 as the target. by doing so, TSLP is expected to be the target gene of mircroRNA-395 and then mediate Th2 differentiation in nasal tissue. After that, we plan to determine this regulatory effect of microRNA-395 on TSLP/Th2 cells in nasal epithelial cell and human bronchial epithelial cells line culture by using microRNA-395 lentivirus vectors and cytokine antagonists, and in mice model. Finally we will screen the possible signal pathways by take advatage of gene chip technology, and confirm the related signal mechanisms using specific inhibitor. Our findings on the regulatory effect of microRNA-395 on TSLP/Th2 subset in allergic rhinitis are expected to be benefical for the improvement clinical efficacy.

国内外许多研究证实，变应性鼻炎是一种以Th2细胞优势分化为特征的疾病，鉴于miRNA被证实参与调控Th2细胞分化，TSLP通过树突状细胞等参与Th2细胞分化的调控，而生物信息学服务提示TSLP是miR-395的作用靶向，因此我们假设变应性鼻炎患者的Th2细胞的优势分化可由miR-395通过TSLP进行调控。为此，本研究拟首先检测变应性鼻炎组织中miRNA差异表达及Th2细胞因子和TSLP水平并进行关联分析和组织定位，确定miR-395和其靶向TSLP，然后通过离体鼻息肉细胞以及人支气管上皮细胞株的培养，再构建miR-395慢病毒载体转染和TSLP拮抗等干预检测其对于CD4+细胞分化的影响，并通过构建成熟的动物疾病模型验证这一分化影响；最后通过基因芯片技术筛选可能的信号通路，通过通路的特征性阻断剂证实相关信号机制，为提高临床治疗的疗效提供依据和新的方法。

变应性鼻炎发病的重要免疫学基础是Th1与Th2细胞之间的失衡，主要表现为Th2细胞优势。而Th2细胞主要分泌相关细胞因子在AR的发病过程中的具有复杂的网络调节作用。通过变应性小鼠模型证实与Th2细胞相关细胞因子水平较正常组普遍升高，其中以IL-4和IL-13较为显著，IL-4和IL-13都可刺激B细胞活化，促进其抗原提呈作用，IL-4和IL-13共享同一受体信号传导系统，同时封阻 IL-4 和 IL-13 生物学活性，将会取得更好的治疗效果。TSLP能激活树突状细胞创建Th2自由微环境，生成Th2细胞因子IL-4，IL-5，IL-13以及大量的TNF-α，在变应性鼻炎小鼠模型鼻甲上用免疫荧光定位到TSLP有大量表达，并且通过荧光定量pcr证实mRNA表达明显高于对照组鼻黏膜，并且在蛋白水平的表达同步上升，通过半定量和定量研究结果都显示TSLPmRNA在变应性鼻炎鼻黏膜中呈高表达。microＲNA参与T细胞发育和功能的调节。通过micRNA实时荧光定量证实在鼻黏膜上miR-375的表达是下降的，与变应性鼻炎模型的细胞因子的表达是呈负相关。利用慢病毒miRNA过表达载体干预变应性鼻炎模型后，通过荧光定量pcr及western blod 检测发现TSLP在鼻粘膜组织中的表达显著下降，同时IL-4和IL-13的含量也显著下调。证实miR-375在TSLP介导的Th2细胞分化中具有明显的调节作用。Th2细胞分化的通路GATA3可能MiRNA-375能通过降低TSLP表达，抑制Th2型细胞的分化的主要信号通路可进一步通过用免疫共沉淀技术或反向阻断的方法明确其信号通路的分子靶点。miＲ-375表达的缺失可会通过直接作用于目的基因TSLP，最终促进Th2 为主的炎症，MiRNA-375能通过降低TSLP表达，抑制Th2型细胞的分化，导致Th2型细胞因子IL-4、IL-13的合成减少，减轻Th2型细胞优势免疫介导的鼻黏膜炎症反应。通过临床病例观察，IL-22阳性表达细胞亚群以及IL-17A阳性表达细胞亚群在AR患者的血清中是显著升高。IL-22和IL-17A的血清水平与病情的严重程度和SIgE水平相关。IL-22和IL-17A在调节变应性鼻炎Th2细胞优势分化引起的炎症反应中起着重要的作用。