shRNA靶向沉默RelB/MyD88诱导的耐受性DC在EAE/MS治疗中的作用及机制

Multiple sclerosis is a common central nervous system demyelinating disease, which is one of the major disease causing young disability and lack of efficient method of specific immunotherapy. We induced tolerogenic dendritic cells (DCs) by 1,25-dihydroxyvitaminD3 in our previous study and found that these tolerogenic dendritic cells had obvious therapeutic effect on experimental autoimmune encephalomyelitis (EAE). Based on our previous studies, we choose RelB and MyD88, which may be the critical intracellular signal transduction molecules inhibited by 1,25-dihydroxyvitaminD3 during the induction of tolerogenic DCs and detect the alteration of these molecules in DCs of EAE and MS patients. To clarify the role of RelB and MyD88 in the pathogenesis of EAE, we will establish EAE animal model in RelB and MyD88 knockout mice. We will induce tolerogenic DCs from EAE and MS patients by silencing the expression of RelB and MyD88 in DCs with short hairpin RNA (shRNA). To investigate whether tolerogenic DCs induced by shRNA have the ability to ameliorate EAE, the tolerogenic DCs are intravenously injected into EAE mice. This study is supposed to provide a new method and the theoretical basis for the treatment of multiple sclerosis.

多发性硬化（MS）是中枢神经系统常见的脱髓鞘疾病，是青壮年致残的主要原因之一，目前尚缺乏有效的特异性免疫治疗手段。我们已经应用1,25-二羟维生素D3诱导了耐受性树突状细胞（DC），回输实验性自身免疫性脑脊髓炎（EAE）后，发现其对EAE有明显的治疗作用。在此基础上，本课题选择1,25-二羟维生素D3诱导耐受性DC过程中可能起关键作用的细胞内信号转导因子RelB和MyD88，研究其在EAE小鼠和MS患者DC中表达水平的变化；应用RelB和MyD88基因敲除鼠诱导EAE模型，研究RelB和MyD88在EAE发病中的作用；应用短发夹RNA（shRNA）沉默EAE小鼠和MS患者DC中RelB和MyD88的表达，诱导耐受性DC，然后将小鼠耐受性DC回输给已发病的EAE，研究其对EAE的治疗作用及机制（创新点），为临床应用耐受性DC特异性治疗MS提供新的方法和理论依据。

多发性硬化（multiple sclerosis, MS）是中枢神经系统（central nervous system, CNS）白质炎性脱髓鞘性疾病。实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis, EAE）是MS的经典动物模型。树突状细胞（dendritic cells, DC）是体内最重要的抗原提呈细胞（antigen presenting cells, APCs），除启动免疫应答外，DC还能促进并维持免疫耐受，可为MS/EAE的特异性治疗寻找新的靶点。.此前，我们应用1,25-二羟维生素D3（1,25-(OH)2D3）诱导耐受性DC（VD3-DC）并回输治疗EAE时能够有效抑制EAE症状。但1,25-(OH)2D3性质极不稳定，VD3-DC对EAE的治疗效果十分短暂，这些缺点限制了其临床应用。本课题中，我们选择1,25-(OH)2D3诱导耐受性DC过程中可能起关键作用的细胞内信号转导因子RelB和MyD88，研究其在EAE DC中表达变化，并应用短发夹RNA（shRNA）分别沉默RelB和MyD88，诱导耐受性DC（RelB/MyD88-shRNA DC），将其回输至EAE，探究其对EAE的治疗作用及机制。我们发现EAE DC中RelB和MyD88的表达显著增加，RelB-shRNA DC、MyD88-shRNA DC和RelB-MyD88-shRNA DC（同时转染RelB-shRNA和MyD88-shRNA的DC）均具有耐受性DC的特征，其表面MHC-II、CD83、CD86的表达明显低于未转染的DC，而PD-L1的表达显著升高。RelB-shRNA DC、MyD88-shRNA DC和RelB-MyD88-shRNA DC均能够显著改善EAE症状并减轻脊髓损伤和炎性细胞浸润。此外，我们发现耐受性DC回输治疗能够减少EAE脾脏内Th1、Th17细胞比例，增加脾脏内调节性T细胞（Treg）和调节性B细胞（Breg）比例。因此我们推断：RelB/MyD88-shRNA DC是通过减弱外周炎症反应的同时提高Treg、Breg的比例从而减少CNS炎症损伤来治疗EAE的。我们的研究探索了诱导耐受性DC的新方法，为临床应用耐受性DC治疗MS提供了新的理论依据。