miR-223对实验性自身免疫脑脊髓炎的调节作用机制研究

MiR-223 is mainly express in bone marrow cell, controlling the differentiation of granulocyte, which plays an important role in cancer and virus infection. Recently study found that miR-223 highly expressed in the T cells of rheumatoid arthritis and human acute leukemia, which has effect on prevention and treatment of the related diseases. But the role of miR-223 in regulating of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis(EAE) mediated by T cells is not clear. Our study found that miR-223 expression significantly increased in the spleen and spinal cord of EAE mice. While, after suppression the expression of miR-223, it can significantly delay the onset time and reduce clinical score. Therefore, our purpose is that miR-223 can promote the development of EAE. The project is proposed to study the regulating role and molecular mechanism of miR-223 on experimental autoimmune encephalomyelitis and related T helper cells differentiation by over expression and inhibition miR-223 expression in stable cell lines and animal model of mice in vivo and in vitro. We aim to reveal the regulation mechanism of miR-223 on EAE mice through the whole-body, cell and molecular level. It will be the foundation for miR-223 to treat human multiple sclerosis and related autoimmune and inflammatory disease.

miR-223主要在骨髓细胞表达，调控粒细胞的分化，在癌症和病毒感染中发挥作用。最近，研究发现miR-223在风湿性关节炎和急性白血病人T细胞中高表达，具有预防和治疗相关疾病的作用。但对T细胞介导的多发性硬化症及其动物模型自身免疫性脑脊髓炎（EAE）的调节作用尚不清楚。我们研究发现EAE小鼠脾脏和脊髓中miR-223表达显著升高，并且干扰抑制miR-223后，可显著延缓发病时间并降低疾病临床评分。因此，我们认为miR-223可促进EAE发生发展。本项目拟通过超表达及抑制miR-223表达的稳定细胞系和疾病动物模型，在体内小鼠EAE疾病模型和体外T细胞定向分化中，研究miR-223对实验性自身免疫性脑脊髓炎及其相关辅助T细胞分化的调节作用和分子机制。在整体、细胞和分子水平揭示miR-223调节EAE的作用机理，为人类多发性硬化症以及miR-223相关的自身免疫和炎症性疾病的预防和治疗奠定

miR-223主要在骨髓细胞表达，调控粒细胞的分化，在癌症和病毒感染中发挥作用。最近，研究发现miR-223在自身免疫关节炎和急性白血病人T细胞中高表达，对有效预防和治疗相关疾病有一定作用。但对T细胞介导的自身免疫性脑髓炎（EAE）的调节作用未见报道。本项目研究发现EAE小鼠脾脏/脊髓中miR-223表达显著升高，并且干扰抑制miR-223后，可显著降低发病率和临床评分。本项目证明miR-223对小鼠EAE病例发生发展具有调节作用，阐明miR-223对T细胞分化和EAE发生的分子机制，探索以miR-223为靶点的自身免疫疾病治疗新途径。.主要研究结果如下：.1.敲低miR-223对小鼠EAE发生具有保护作用。我们构建敲低miR-223的慢病毒，MOG抗原诱导小鼠EAE模型，发现注射miR-223慢病毒组小鼠发病明显减缓，临床评分降低，脊髓脱髓鞘和炎性细胞浸润程度显著低于对照组。说明敲低miR-223对小鼠EAE发生、脊髓脱髓鞘和中枢神经系统炎症反应具有保护作用。.2.敲低miR-223对EAE小鼠体内不同T细胞亚群分化具有重要调节作用。流式细胞术检测小鼠脾脏、淋巴结、中枢神经系统，敲低miR-223显著降低脾脏中Th1和Th17细胞转录因子和相关细胞因子T-bet、IFN-γ、IL-17A等表达；实施定量检测脾脏中这些因子表达，同样发现敲低miR-223降低IL-17A和IFN-γ基因mRNA表达水平。.3.敲低miR-223抑制Th1和Th17细胞体外分化。体外分离小鼠脾脏初始T细胞，诱导分化为Th1和Th17细胞，进一步验证抑制miR-223可以显著抑制Th17细胞的分化，降低T-bet表达，降低致病性Th17细胞分化。但是病理反应机制、具体靶点不清楚，本项目随后就miR-223的具体靶点和机制进行详细研究。.4.确定miR-223靶基因。通过萤光素酶报告基因分析、实时定量和WB蛋白分析，确定Hsp72为miR-223的靶基因。.5.寻找miR-223下游起作用基因。再次验证，抑制miR-223后，EAE小鼠的CNS系统中Stat3蛋白磷酸化水平显著降低，限制Th17细胞分化。.因此，我们认为miR-223可促进EAE发生发展。本项目在整体、细胞和分子水平揭示miR-223调节EAE的作用和细胞及分子机制，为与miR-223相关的自身免疫疾病的预防和治疗奠定基础