2015-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656949摘要：毛毛樣腦血管疾病（Moyamoya disease，MMD)為一罕見的腦血管疾病， 與兩侧的内頸動脈（internal carotid arteries, ICA )以及顧内動脈之閉塞或狹窄所 誘發的小血管大量代償並漸進性的雜亂無章之成長有關。其好發年齡有兩極化的 現象：兒童時期發病者，大多是因為腦部血管狹窄所導致的血流不足，病徵多為 缺血性中風；成年期才發病者，則多見腦内侧枝血管破裂，導致顧内出血性中風。 此疾病主要盛行於東亞地區，包含日本、韓國及中國等區域，具亞洲血統、手足 中有罹病者，特別是同卵雙胞間存在更高的罹病風險，由此推論基因的差異性可 能對疾病的發生扮演重要角色。依照基因連鎖分析有伍個可能的基因位置與 MMD之致病具有相關性，其中僅有RNF213反基因突變被證實會導致 MMD。我們過去已完成三十六個MMD患者之基因位點分析，並發現 十一個新的RNF213造成胺基酸序列之錯義突變(missense mutation)，在剩餘的二 十五個家族中，則未有JCC42基因突變發生。MMD的病理特徵為血管内膜的增厚與纖維母細胞的增生，然而RNF213於 疾病發生的過程之可能參與之角色仍無定論。由於nitric oxide (NO)及heme oxygenase-1 (HO -1)可能在血管損傷時，與内膜的增厚與纖維母細胞增生扮演重 要角色，我們嘗試抑制BJ纖維母細胞及人類臍帶血管内皮細胞(HUVEC)之 ACTA2反RNF213之基因表現，目前結果顯示BJ細胞之/7VCW mRNA顯著下降 而m-1上升;於HUVEC則幾乎觀察不到/+7VOS的表現，然而其eTVCW則有顯 著增加之現象。這些變化暗示，這些基因可能參與MMD的致病機制。因此，本研究首先希望了解RNF213及ACTA2的生理功能，及其突變於 MMD所扮演的角色。這部分我們將使用免疫沉澱搭配蛋白質體的分析方法，去 搜尋可能與RNF213不同突變型結合之蛋白質；並搭配iTRAQMASS分析病人 血漿中蛋白質的差異性，以發掘參與於MMD致病過程之未知蛋白質。其次將於 BJ與HUVEC細胞中抑制RNF213及ACTA2基因的表現，以觀察在 haploinsufticiency的情境下，這些基因的功能與纖維母細胞增生及血管内膜增厚 的相關性。這些結果也將由轉殖入錯義突變之細胞以及由病人血液所製備之淋巴 母細胞株(lymphoblast cell line)進行驗證。最後，我們將利用次世代基因定序(NGS) 平台找出其餘可能與疾病有顯著相關性之基因。期望經由本計晝，可以建立 MMD相關可能致病之分子機轉之脈絡，這些結果我們並將使用BJ及HUYEC 之共培養和HUVEC條件培養基(conditioned media)的方式，以釐清細胞間互相影 響的作用方式，是否與導致MMD之症狀相關。<br> Abstract: Moyamoya disease (MMD) is a rare steno-occlusive disease involving bilateral internal carotid arteries (ICA) and also intracranial arteries which induces massive progression of small collateral vessels. The age of disease onset is in a bimodal distribution. Cerebrovascular occlusion-induced infarction in MMD patients usually occurs at young age; however, an intracerebral bleeding from the fragile collateral vessels is more common in adults. The prevalence rate is high in east Asian countries, especially in Japan, Korea and China. High occurrence of MMD in siblings and twins as well as the clustering in Asian ethnicity indicate a genetic predisposition factor in prevalence. There are five linked loci for MMD with 2 known causative genes, RNF213 and ACTA2 gene. In our previous study, we recruited 36 MMD families and identified 11 missense mutations in RNF213 gene.The pathological hallmarks for MMD are intimal thickening (IT) and fibroblast proliferation (FP) of the moyamoya vessels. It remains elusive how the mutations of RNF213 result in the pathological change in ICAs. Growing evidences demonstrated that nitric oxide (NO) and heme oxygenase-1 (HO -1) play an intriguing role in the pathogenesis of IT and FP in balloon damaged vessels. Thus, we evaluated the levels of NO synthase and HO -1 in the BJ fibroblasts and HUVEC cells transfected with siRNF213 and siACTA2. In fibroblasts, the miRNA level of iNOS was reduced markedly with increase expression of HO -1. On the contrary, the level of iNOS was nearly absent, but the level of eNOS increased markedly.Based on the preliminary results, we proposed three major aims for this project. The first one would be to explore the basic cellular function of RNF213 and ACTA2 genes and its derangement associated with the mutations in these two genes. To identify the associated protein(s) of RNF213, immunoprecipitation would be carried out followed by MASS analysis. Furthermore, we would also evaluate any change of circulatory protein(s) in patient’s plasma using the iTRAQ MASS proteomic analysis. Secondly, we would validate the newly identified protein(s) which shows associated with RNF213 and investigate the molecular mechanisms leading to IT and FP. In addition to the knockdown cell models, the knockin missense mutation models by gene editing and the human lymphoblast cell line will be constructed and employed for further functional research. In addition to these two genes, we would use next generation sequencing platform to identify the unknown gene for MMD. We would try to construct a functional integration map of the molecular mechanism leading to MMD. Finally, as the unmatched results from fibroblast and endothelial cells, a co-culturesystems or conditioned media administration will be employed to elucidate any molecular cross-talk between these two cells in the pathogenesis of MMD.毛毛樣腦血管疾病内膜的增厚纖維母細胞增生RNF213ACTA2基因突變Moyamoya diseaseintimal thickeningfibroblast proliferationRNF213ACTA2mutation