利用斑马鱼模型研究白细胞介素7受体在髓鞘脱失和形成中的作用机制

The myelin sheath is the membrane structure protecting, supporting and nourishing axons. Demyelinating diseases are a variety of diseases in which the neuronal myelin sheath is damaged due to genetic, environmental factors or unknown factors. The myelin structure, myelin synthesis and gene expression are highly conserved between zebrafish and mammals. Therefore, zebrafish is an ideal model to study the demyelination and myelination. In the previous study, we generated a heritable and stable transgenic line Tg (mbp:nfsB-egfp). Treatment of Tg (mbp:nfsB-egfp) larvae with metronidazole (Mtz) resulted in the selective ablation of oligodendrocytes and led to demyelination. Array analysis showed that interleukin 7 receptor (il7r) gene was strongly downregulated during this demyelination. However, gaps still remain regarding the role of il7r during demyelination. Our aim is to test the hypothesis that il7r regulates the demyelination or myelination. Four sub aims will be undertaken: (1) the expression of il7r, morphology, imaging and behaviors will be tested on an adult demyelinating model; (2) the expression of molecules or genes in il7r related pathways will be tested to characterize the mechanisms of il7r down-regulation; (3) the effects of il7r in embryonic myelination will be studied by the targeted knockdown of il7r with morpholino oligonucleotides and (4) il7r knockout mutant zebrafish will be generated to investigate the role of il7r on the differentiation of oligodendrocytes. The studies proposed here will contribute to the comprehensive understanding of il7r in demyelination and myelination and will help develop therapies for il7r-based treatment of demyelinating diseases in humans.

髓鞘是保护、支持轴突的膜性结构，在遗传和/或环境因素作用下受到损伤，可引起以多发性硬化为代表的脱髓鞘疾病，目前尚无有效治疗。斑马鱼是研究髓鞘脱失和髓鞘形成的理想动物模型，前期构建的转基因斑马鱼Tg (mbp:nfsB-egfp)可在甲硝唑诱导下发生特异性髓鞘脱失，基因芯片表达谱分析发现白细胞介素7受体（il7r）表达下调。本项目在此基础上，以斑马鱼为模型深入研究il7r在髓鞘脱失与形成的作用。研究工作包括四部分：第一，成体鱼髓鞘脱失的影像学和运动功能改变以及il7r在少突胶质细胞的表达；第二，il7r在特异性髓鞘脱失中的作用途径与机制；第三，通过对胚胎斑马鱼敲减il7r基因研究其在髓鞘形成的作用；第四，构建il7r基因敲除的斑马鱼突变体，探究il7r与少突胶质细胞分化的关系。上述工作将进一步阐明髓鞘脱失的发病机制，并为开发以il7r为靶点治疗多发性硬化等脱髓鞘疾病提供理论依据。

髓鞘是保护、支持轴突的膜性结构，在多种因素作用下受到损伤，可引起脱髓鞘疾病，目前缺乏有效治疗。转基因斑马鱼Tg(mbp:nfsB-egfp)是研究髓鞘脱失和髓鞘形成的理想动物模型。本项目主要进行了以下四方面研究工作。第一，白细胞介素7受体（il7r）在斑马鱼髓鞘脱失与形成中的作用及机制。在Tg(mbp:nfsB-egfp)转基因斑马鱼诱导髓鞘脱失，以磁共振成像技术进行扫描，表达谱芯片筛选差异表达基因，发现il7r显著下调，并通过JAK/STAT途径增加细胞凋亡。上述工作发现了il7r是调控髓鞘脱失和髓鞘形成的关键分子。第二，白细胞介素7受体调控神经细胞生成的作用及机制。利用CRISPR/Cas9技术制备il7r基因缺失型斑马鱼突变体，il7r-/-呈现小眼球等表型改变，视网膜神经细胞发育延迟，视觉功能受损，il7r基因缺失后光传导信号通路相关的基因显著下调。上述工作发现了il7r具有调控神经细胞生成的新功能。第三，环氧合酶-2（COX-2）抑制剂塞来昔布通过改善免疫和炎性微环境促进髓鞘再生。以塞来昔布治疗髓鞘脱失，观察髓鞘再生并探究其机制。研究发现，塞来昔布具有较低的体内毒性，可通过靶向抑制COX-2改善炎性微环境，抑制少突胶质细胞凋亡，增强内源性髓鞘再生，这为脱髓鞘疾病的治疗提供了潜在的药物策略。第四，甲泼尼龙纳米载药系统的构建及对视网膜感光细胞损伤的治疗应用。以锆-纳米卟啉金属有机骨架（Zr-NPMOF）吸附甲波尼龙（MPS），构建MPS-NPMOF新型纳米载药系统，经眼内注射递送到感光细胞损伤区。结果表明，NPMOFs低毒、成像能力强，是一种理想的骨架材料，MPS-NPMOF眼内注射实现了药物缓释，促进感光细胞再生。项目资助期内，累计在SCI收录期刊发表学术论文8篇；获得国家发明专利授权1项，申请国家发明专利1项；3人获博士学位，5人获硕士学位。