microRNAs在实验性哮喘中对CD4+ T细胞的表观遗传的调节机理研究

Dust mite allergen Der f1 is one of the important airborne allergens for asthma. Asthmatic murine induced by dust mite allergens provides an ideal experimental model for analyzing the pathogenesis of asthma and its specific immunotherapy. CD4+ T cell subsets, especially the imbalance of Th1 / Th2 cells, play the crucial role in asthmatic pathogenesis. It might be an effective way to treat allergic asthma through rectifying the proliferation and differentiation of CD4+ T cell subsets. MicroRNAs (miRNAs) are important molecules in the proliferation and differentiation of CD4+ T cells, but its epigenetic regulation in the proliferation and differentiation of CD4+ T cell remains to be further studied in the pathogenesis of asthma. Twenty miRNAs were found, which located in the 3 kb nearby the up- or down-stream of differential DNA peaks, after analyzing methylated DNA data. This project intends to analyze miRNA expression profiling of CD4+ T cells from the spleen of murine asthmatic model. After integrating the data with methylated DNA data. Functional assay of T cell is performed to identify the effects of differential miRNAs in the proliferation and differentiation of CD4+ T cell from murine asthmatic model. Their epigenetic mechanisms are also analyzed using mRNA expression profiling. It will provide experimental evidence for focus on miRNAs in the pathogenesis of asthma and provide new ideas for the specific treatment of asthma.

粉尘螨Der f1变应原是引起哮喘的重要气传变应原之一。螨性哮喘小鼠为哮喘发病机制及治疗研究提供了理想模型。CD4+ T细胞亚群Th1/Th2细胞失衡处于哮喘发病的中心环节，改善其增殖及分化可能是治疗哮喘的有效途径。微小RNAs（miRNAs）是调控CD4+ T细胞增殖和分化的重要分子，但其对哮喘CD4+ T细胞增殖分化的表观遗传调控尚不清楚。CD4+ T细胞DNA甲基化数据分析发现20个miRNAs位于DNA甲基化差异峰值上下游3 kb附近。本课题拟分析哮喘小鼠脾CD4+ T细胞的miRNA表达谱，并与DNA甲基化数据整合分析。T细胞功能实验验证差异miRNAs对哮喘CD4+ T细胞增殖分化的影响，并整合差异mRNA表达谱数据，分析差异miRNAs对哮喘发病过程中对CD4+ T细胞增殖和分化的表观遗传调控机制。以期为清晰miRNAs在哮喘发病中的意义提供实验依据及为治疗哮喘提供新思路。

项目背景.过敏性哮喘是临床常见的季节性多发病，目前尚无特异性治疗方法。CD4+ T细胞亚型Th1/Th2/Treg/Th17比例失衡为其主要致病因素。非编码RNAs参与了CD4+ T细胞的增殖与分化。但其详细分子机理尤其是在表观遗传调控有待阐明。.主要内容.（1）根据上一项目获得的差异lncRNAs分子，通过GEO数据集进行筛选，确定了linc00619分子作为靶分子，对其在肺上皮细胞中的增殖功能和机制进行了探讨。（2）对CD4+ T细胞分化的关键分子PKCθ（上一项目筛选获得）进行了基因敲除小鼠C57BL/6的构建，分别制备了急性哮喘（21d）和慢性哮喘（42d）动物模型，流式细胞术分选CD4+ T细胞，DNA甲基化检测筛选差异表达基因和后续的生物信息学分析。也对该基因在胰岛素抵抗方面进行了初步研究。（3）探索了草乌甲素在过敏性哮喘炎症中的治疗作用及其机制探索。同时也对醇质体等药物透皮递送的改进和效果进行了一系列的探索。（4）对其他一些ncRNA分子（包括lncRNAs和miRNAs）的功能和机理进行初步探讨。.重要结果.（1）FISH显示linc00619定位于细胞质。linc00619敲除促进了BEAS-2B的增殖和侵袭，共得到3461个差异mRNAs，Western blot证实，linc00619敲除活化了AKT和ERK。（2）PKCθ敲除后甲基化区域动态改变，共获得了2659个差异基因启动子（高、中、低CpG启动子区域）和1987个差异CpG岛（基因间、基因内以及启动子去）；此外，PKCθ敲除抑制了胰岛素分泌和耐受。（3）草乌甲素可抑制PKC-δ/NF-κB信号缓解OVA诱发的气道炎症；细胞实验表明，可抑制TLR4的表达以及NF-κB和MAPK的活化减少炎症因子的释放。醇质体递送系统可有效激发DC细胞的活化并增强免疫应答。.科学意义.初步观察了PKCθ在哮喘中的详细作用及表观调控；尝试了哮喘透皮给药的治疗效果；探索了其他ncRNAs的分子功能，为后续哮喘的治疗提供了实验数据。