烟雾病易感基因RNF213调控内皮祖细胞增殖、迁移及分化的机制研究

我们发表的荟萃分析表明，RNF213基因突变与烟雾病（MMD）的高发病风险相关。前期研究已证实敲除斑马鱼rnf213a基因可出现颅内动脉血管壁狭窄和芽生血管形成，且芽生血管的内皮细胞形态异常等MMD血管病变特征，而该基因ATP酶区和环指结构缺陷所致的血管病变程度较轻。为进一步探索RNF213 重要突变位点p.R4810K在烟雾病异常血管形成中的作用，本研究拟利用流式细胞术、免疫荧光、分子克隆、免疫共沉淀等技术分析p.R4810K突变对内皮祖细胞（内皮细胞的前体）增殖、迁移和分化的影响，以及干扰素和缺氧对该影响的促进作用。利用CRISPR/cas9技术构建rnf213a p.R4813K（RNF213 p.R4810K同源）转基因斑马鱼，活体观察该突变点对异常血管壁中内皮细胞和平滑肌细胞的影响，完善MMD的发病机制，为药物研制与筛选提供重要的靶位点。

Our published meta-analysis showed RNF213 gene variations were associated wtih the high risk of moyamoya disease(MMD).Our previous experiments found that knocked out the rnf213a gene in zebrafish could lead to intracranial arterial wall trunk irregular stenosis and abnormalities sprouting angiogenesis,and the abnormal morphology of endothelial cells in sprouting vessels, which was consistent with the characteristics of vascular lesions in MMD. It has been proved the vascular lesions were lesser in zebrafish knockouted ATP enzyme region and the ring finger domain of rnf213a.To further explore the formation mechanisms of abnormal vessels in MMD by the important variation p.R4810K, we analyse the effcet of RNF213 p.R4810K regulating the proliferation, migration and differentiation of endothelial progenitor cells using flow cytometry, immunofluorescence, molecular cloning and co-immunoprecipitation, and demonstrate the promting effect of interferon and hypoxia.We want to develop a rnf213a p.R4813K (RNF213 p.R4810K homologous) transgenic zebrafish with CRISPR /cas9 technology to observe the dynamic changes of vascular endothelial cells and smooth muscle cells in vivo and perfect the pathogenesis of MMD in order to provide an important target site for drug development and selection.