家族性烟雾病诱导多能干细胞模型构建及其发病机制研究

Moyamoya disease（MMD） is a common cause of stroke in children and young adults in china. Its characteristic is the appearance of abnormal vascular network in the circle of Willis. The results of our previous studies confirmed that ring finger protein 213 gene (RNF213) is the major susceptibility gene for Chinese MMD patients. Plenty of studies have been conducted to explore the pathogenesis of MMD by endothelial cells, RNF213 knockdown zebrafish model and mice model. However, the role of RNF213 mutation in pathogenesis of MMD is still unclear. The heterogeneous nature of MMD and lack of proper animal models made it quite chanlleging to investigate the pathogenesis. Moreover, it is also difficult to obtain the specimen of moyamoya vessels. Our previous studies on cytokines, circular RNA and long non-coding RNA of MMD patients showed that Akt pathway may involve in the cascades of angiogenesis of MMD. Therefore, we will collect samples from familial MMD patients with RNF213 p.R4810K or p.K4160Q mutation and their familial numbers. Moreover, we will apply induced pluripotent stem cells (iPSCs) technology to generate endothelial cells derived from MMD and RNF213 mutation-specific iPSCs. By iPSCs and iPSECs models, we will study the effects of RNF213 mutation on cellular functions and Akt pathway. Furthermore, screening for predictive cytokines and analysis the correlations between these cytokines and clinical phenotype and surgical outcomes of MMD patients will be performed. Our study may help to clarify the pathogenesis of moyamoya disease and provide new targets for disease treatment.

烟雾病是我国儿童和青壮年卒中常见病因，其特征性表现为脑底异常血管网形成。我们前期研究提示RNF213是中国烟雾病患者的主要易感基因。但对内皮细胞、小鼠和斑马鱼等的研究，均未阐明RNF213突变影响血管生成和导致烟雾病发病的机制。该领域研究的瓶颈是无法对病变脑血管直接研究及缺少疾病模型。我们前期基于患者外周血细胞因子、环状RNA和长链非编码RNA的研究提示Akt通路可能调节相关细胞因子，参与烟雾病发病。因而本项目选取RNF213p.R4810K突变和p.K4160Q突变（申请人发现）家系，采用诱导多功能干细胞、基因编辑和免疫组化等技术，1.构建不同基因型iPSCs和iPSECs模型；2.在细胞和分子水平研究RNF213突变对内皮细胞血管生成和Akt通路的影响机制；3.筛选RNF213突变影响的细胞因子，与患者表型和预后进行关联分析。本项目可能发现烟雾病发病的关键机制，为疾病治疗提供新的靶点。

烟雾病是我国儿童和青壮年卒中常见病因，其特征性表现为脑底异常血管网形成。RNF213是中国烟雾病患者的主要易感基因，但对内皮细胞、小鼠和斑马鱼等的研究，均未阐明RNF213突变导致烟雾病发病的机制。该领域研究的瓶颈是无法对病变脑血管直接研究及缺少疾病模型。在本课题中，①我们构建了RNF213突变（p.R4810K）烟雾病iPSCs和iPSECs模型，为研究烟雾病的发病机制提供模型。结果显示携带p.R4810K 突变iPSECs的血管形成能力弱于正常iPSECs，提示RNF213p.R4810K可能通过影响血管形成能力参与烟雾病的疾病发生过程；②通过MMD患者和健康对照的外周血转录组测序结果提示RNF213突变参与血管生成和Akt信号通路调控；③通过分析RNF213突变患者烟雾血管（LSA，TTA，TPA，AChoA，PChoA）的血管造影特征，表明位于羧基末端区域的罕见RNF213突变促进了烟雾病中的动脉生成，影响了疾病严重程度。本课题从基因、分子、细胞、患者水平全面阐述烟雾病的致病机理，为烟雾病的防治和药物筛选提供新的思路和靶点。

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