miR-122对Th17/Treg分化和功能的影响及其在银屑病发病中的作用机制

At present, the etiology of psoriasis and pathogenesis have not been fully elucidated yet, and abnormal activation and differentiation of CD4+ T cells play an important role in the pathogenesis of psoriasis. We screened and verified that the expression of miR-122 is elevated in psoriatic lesions, and Bach2 is a downstream target gene of miR-122, through microRNA microarray and microRNA expression profile microarray in psoriatic lesions and healthy skin. Recent study found that Bach2 gene， a key regulatory factor with wide immune activity , can limit the differentiation of multiple kinds of effector CD4+ T cells and promote the formation of Treg cells effectively to maintain the stability of the immune system and prevent autoimmune inflammatory response. Thus we propose the hypothesis that the elevated expression of miR-122 in psoriatic lesions can affect Th17/Treg differentiation and function by regulating the expression of Bach2 gene and break the Th17/Treg balance resulting in the advantage of Th17 which secret large amounts of IL-17A, IL-17F and IL-22 and promote the development and progression of psoriasis. In this study, both in vivo and in vitro experiments such as cell culture , siRNA transfection, animal experiments and other methods will be carried out to verify this hypothesis to lay the foundation for in-depth study of the immune response abnormalities in psoriasis.

目前银屑病的病因和发病机制尚未完全阐明，CD4+T细胞的异常活化及分化在银屑病的发病中起重要作用。我们在银屑病皮损与健康对照皮肤中通过microRNA芯片与mRNA表达谱芯片技术，筛选并验证了miR-122在银屑病皮损中高表达，Bach2为其下游靶基因。最新研究发现，Bach2基因作为一种具有广泛免疫活性的关键性调节因子，通过限制多种效应性CD4+T细胞的分化，有效促进Treg细胞的形成，维持免疫系统的稳定，阻止自身免疫炎症反应的发生。由此提出科学假说：银屑病皮损中高表达的miR-122可通过调控Bach2基因的表达，影响Th17/Treg的分化和功能，打破Th17/Treg平衡，出现Th17优势反应，分泌大量的IL-17A、IL-17F和IL-22，促进银屑病的发生发展。本研究将采用细胞培养、siRNA转染、动物实验等方法进行体内外实验验证该假说，为深入研究银屑病的免疫反应异常奠定基础。

目的：银屑病是一种常见的免疫介导的慢性炎症性皮肤病。目前银屑病的病因和发病机制尚未完全阐明，CD4+T淋巴细胞的异常活化及分化在银屑病的发病中起重要作用。据报道Th17/Treg免疫失衡有助于银屑病的发生发展。本研究的目的是探索Bach2基因对Th17/Treg细胞分化和功能的影响及其在银屑病发病中的作用机制。.方法：从健康人外周血中磁珠分选出初始CD4+T淋巴细胞，然后在体外不同的极化条件下使其诱导分化成Th17或Treg细胞。体外转染Bach2-siRNA或miR-144-3p观察其对Th17/Treg细胞分化和功能的影响。流式细胞术检测Th17细胞、Treg细胞的数量。RT-PCR检测其特异性转录因子mRNA水平的变化。ELISA 法检测培养上清液中 IL-17、IL-22和IL-10细胞因子的含量。生物信息学预测和双荧光素酶报告实验确定miR-144-3p和Bach2基因的靶向关系。.结果：我们发现与健康对照者相比，银屑病患者的外周血中CD4+ T淋巴细胞中Bach2 mRNA显著下调。在人外周血CD4+初始T细胞向Th17细胞诱导分化的过程中，敲减Bach2基因后，我们发现诱导的Th17细胞（CD4+IL17+）的百分比增加; Th17特异性转录因子视黄酸相关孤儿受体γt（RORγt）的mRNA表达增加。同时ELISA法检测培养上清液中 IL-17、IL-22细胞因子的含量增加。相反，在人外周血CD4+初始T细胞向Treg细胞诱导分化过程中，敲低Bach2基因后，发现诱导的Treg细胞（CD25+FOXP3+）的百分比降低; Treg特异性转录因子叉状头转录蛋白3（FOXP3）的mRNA表达也降低。随后通过microRNA芯片技术发现miR-144-3p在银屑病外周血CD4+T细胞中高表达。双荧光素酶报告基因检测实验证实Bach2为其下游靶基因。体内外实验结果显示，过表达miR-144-3p，诱导分化的Th17细胞比例增加，Treg细胞比例及功能下降；下调miR-144-3p后得到相反的结果。.结论：银屑病外周血中高表达的miR-144-3p可能通过调控Bach2基因的表达打破Th17/Treg平衡，出现Th17优势反应，分泌大量的IL-17A、和IL-22，从而促进银屑病的发生发展。