长链非编码RNA NRON对自身免疫脑脊髓炎的调控作用机制研究

Multiple sclerosis (MS) is an inflammatory demyelination disease of the central nervous system, its pathogenesis is not fully understood. But it was believed that MS was a Th1 and Th17 cell-mediated autoimmune diseases and there are no ideal treatments for MS so far due to it is ease relapse after treatment. Experimental autoimmune encephalomyelitis (EAE) is an animal model which mimics the clinical course and pathological symptoms of MS, and is an important model for the basic study of autoimmune inflammation. There are no reports about lncRNA (long-noncoding RNA) function in pathogenesis of MS / EAE. NRON is a human-mouse conserved lncRNA which was able to inhibit T-cell activation. Our preliminary data shown that interference suppression NRON by lenti-virus can significantly increased the clinical scores and incidence of EAE, and these was associated with promoting Th1 and Th17 cells responses and spinal cord inflammation in EAE. Base on these findings, the present proposal will investigate the regulatory function of NRON in pathogenesis, demyelination and inflammation of central nervous system of EAE, by using mouse EAE model and in vitro helper T cells differentiation model; in vivo and in vitro study the roles and molecular mechanisms of NRON in regulating the differentiation and balance of Th1, Th2, Th17 and Treg cells type. Overall, this proposal will reveal the cellular and molecular mechanisms of NRON in regulation of pathogenesis of EAE/MS, and results of this study will useful for better understanding the pathogenesis of MS/EAE and other autoimmune inflammatory diseases, it will open new avenues for treatment of these diseases.

多发性硬化症（MS）是中枢神经系统炎症性脱髓鞘疾病，其发病机制尚不完全清楚，但多认为是Th1和Th17细胞介导的自身免疫疾病，治疗后会缓解复发，很难治愈。实验性自身免疫性脑髓炎（EAE）是MS的动物模型，也是自身免疫炎症研究的重要模型。有关lncRNA在MS/EAE中的研究尚属空白。NRON是人鼠保守的lncRNA，可抑制T细胞激活。我们发现干扰抑制NRON后，可显著增加EAE临床评分和发病率,促进EAE中Th1和Th17细胞分化及脊髓炎症。本项目拟通过小鼠EAE模型和辅助T细胞分化模型，慢病毒干扰NRON后详细研究NRON对EAE发生、脊髓炎症和脱髓鞘的调控作用；体内和体外研究其对EAE中Th1、Th2、Th17和Treg细胞分化平衡的调控作用和分子机制。在整体、细胞和分子水平揭示NRON调节EAE病理发生的细胞和分子机制，为认识MS/EAE等自身免疫炎症疾病的病理发生及其治疗开辟新途。

长链非编码RNA(lncRNA)可调控多种疾病，是近年研究热点。NRON是人鼠保守的lncRNA，可结合抑制T细胞转录因子NFAT进而抑制T细胞激活。最近研究显示NRON对心血管疾病和肠炎均具有调控作用，可有效治疗相关疾病。但NRON对T细胞介导的人多发性硬化症（MS）及其动物模型自身免疫性脑髓炎（EAE）的调节作用尚不清楚。我们研究发现干扰抑制NRON后，可显著增加EAE发病率和临床评分，促进EAE中Th1和Th17细胞分化及脊髓炎症反应。本项目通体内小鼠EAE模型和体外各种T细胞亚型分化模型，经慢病毒干扰NRON后，详细研究NRON对EAE病理发生的调控作用及细胞和分子机制，研究其对EAE中Th1和Th17等T细胞亚型分化平衡的调节作用和分子机制。在整体、细胞和分子水平揭示NRON调节EAE的作用机理，为NRON相关的MS/EAE等自身免疫炎症疾病的预防和治疗开辟新途径。.主要研究结果如下：.1. 敲低NRON对小鼠EAE发生具有促进作用。我们构建敲低NRON的慢病毒，MOG抗原诱导小鼠EAE模型，发现注射NRON慢病毒组小鼠发病增强，临床评分升高，脊髓脱髓鞘和炎性细胞浸润程度显著高于对照组。说明敲低NRON对小鼠EAE发生、脊髓脱髓鞘和中枢神经系统炎症反应具有促进作用。.2. 敲低NRON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，敲低NRON显著增加脾脏中Th1和Th17细胞相关细胞因子IFN-γ、IL-17A表达。.3. 敲低NRON促进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NRON可以显著促进Th1和Th17细胞的分化。但是病理反应机制、具体靶点不清楚，本项目随后就NRON的具体作用机制进行详细研究。.4. 寻找NRON和下游起作用基因。再次验证，抑制NRON后，EL4细胞中NFATC2表达增加，促进入核。同样增加了GSK-3β和P65蛋白磷酸化水平，促进Th17细胞分化。.因此，我们认为NRON可抑制EAE发生发展。本项目在整体、细胞和分子水平揭示NRON调节EAE的作用和细胞及分子机制，为与NRON相关的自身免疫疾病的预防和治疗奠定基础。本项目中NRON抗炎的生理过程与多种疾病相关，具有广泛的医学应用价值。