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研究生:丁鶴婷
研究生(外文):He-Ting Ting
論文名稱:調查受干擾的免疫細胞對法布瑞氏症發病機制的影響
論文名稱(外文):Investigate the contribution of disturbed immune cell in the pathogenesis of Fabry disease
指導教授:牛道明徐嘉琳徐嘉琳引用關係
指導教授(外文):Dau-Ming NiuChia-lin Hsu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:80
中文關鍵詞:法布瑞氏症溶酶體巨噬細胞
外文關鍵詞:Fabry diseaseLysosomeMacrophage
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法布瑞氏症(Fabry disease)是一種性聯遺傳的溶酶體堆積症,由於 α-galactosidase A (GLA) 的基因缺陷,導致鞘醣脂(glycosphingolipids, GSLs)的代謝中間產物 globotriaosylceramide (Gb3) 無法被分解,而累積在全身細胞內的溶酶體中,造成細胞的損害,進一步導致心臟、腎臟、血管或神經方面的病變。過去有研究指出,法布瑞氏症病人體內某些免疫發炎因子的表現量有高於一般正常人的趨勢,但法布瑞氏症與免疫反應之間的關係尚未被解釋清楚,因此我們想更進一步了解當 Gb3 不正常累積在免疫細胞時,對免疫細胞及其下游的免疫反應是否會造成影響,甚至影響病徵的表現。
在本研究中,我們針對幾個主要的免疫細胞族群,分析細胞中 GLA 基因的表現,發現單核球有最高的 α-GLA 表現量,同時也觀察到法布瑞氏症病人的單核球中,Gb3有顯著較高量的累積,說明這個免疫吞噬細胞族群可能在鞘醣脂的代謝過程中扮演重要的角色。進一步利用法布瑞氏症小鼠模型 (B6;129-Glatm1Kul/J) 和正常老鼠做比較,觀察在免疫細胞數量上或由骨髓幹細胞分化的巨噬細胞功能上,是否存在差異。同時,我們也利用Crispr技術建立將 GLA 基因剔除的人類單核球的 THP-1 細胞株,進一步刺激使之分化成巨噬細胞,觀察人類細胞和老鼠細胞實驗的結果是否有相同趨勢。

目前法布瑞氏症治療辦法是長期將合成酵素施打在病人身上,並且配合使用抗發炎藥物來緩和病程。我們在免疫系統上的研究結果將能夠提供更專一及完善設計的療程,來幫助法布瑞氏症患者有更好的疾病管理。

Fabry disease (FD) is an X-linked lysosome storage disease resulted from the mutations of α-galactosidase A (GLA). GLA participates in the metabolism of glycosphingolipids (GSLs), and is responsible for the breakdown of globotriaosylceramide (Gb3). The detective GLA function leads the failure of Gb3 degradation and its accumulation in the lysosome. This abnormal accumulation of Gb3 has been linked to cell or tissue damage, and further leads to heart, kidney, and vascular or neurological dysfunctions. Previous reports observed that the certain cytokines levels in the FD patients are higher than normal people, without clear pathophysiology. We aim to investigate how GLA mutation may influence immune cell functions and immune responses in FD patients.
To understand which immune cell population may receive the biggest impact in the absence of GLA, we first analyzed the GLA expression in major immune cell populations in peripheral blood. We found that monocyte had the highest expression of GLA, both in transcriptional and translational levels. Moreover, we also observed significant amount of Gb3 accumulation in the FD patient monocytes. Together, these results suggested that the monocytic / phagocytic lineage of cells may play crucial roles in the GSLs metabolism, and also implied their vulnerability upon GLA deficiency. To further understand the impact of GLA deficiency on immune system, we examined the immune cell distributions and members in different ages of GLA knockout animals. We also generated bone marrow derived macrophage (BMDM) to further investigate the functional impact of GLA deficiency on phagocytic immune cells. To correlate our observations in murine cells, we used CRISPR technique to establish the GLA knockout human monocytic cell line (THP-1) and used it as a model system for functional studies.
Currently, the treatment of FD is regular intravenous injection with recombinant enzymes. Our results may provide the insights on the immune system of FD patients and facilitate the development of new treatment regimen.

誌謝 ……………………………………………………………………………………….…. i
中文摘要 ……………………………………………………………………………….……. ii
英文摘要 …………………………………………………………………………….……….. iii
目錄 …………………………………………………………………………...……..……... v
圖表目錄 ………………………………………………………………………….………... vi
第一章論 ……………………………………………………………………………..…...…. 1
1.1溶酶體儲積症 ………………………………………………………………….………... 1
1.2 法布瑞氏症 …………………………………………………............…….......……….. 2
1.3 法布瑞氏症的疾病症狀及分類 ………………………………………….…...….….… 3
1.4 疾病的診斷與治療 …………………………………………………………...………… 4
1.5 法布瑞氏症的器官組織纖維化 ……………………………………………...…..….… 5
1.6 巨噬細胞的分類及功能 …………………………………………………………......… 7
第二章 研究動機 ………………………………………………………………….…..…... 9
第三章 實驗材料與方法 ……………………………………………………….………......... 11
3.1 人類細胞相關實驗 ……………………………………………………….………............. 11
3.2 老鼠細胞相關實驗 ……………………………………………………….………............. 18
3.2 細胞株相關實驗 ……………………………………………………….………................ 25
第四章 實驗結果 ……………………………………………………………….……....... 32
4.1 主要免疫細胞群的GLA基因表現量分析 ……………………………….………..… 32
4.2 建立法布瑞氏症的老鼠模型或人類細胞株 ……..…….....................……...…….... 32
4.3 Gb3累積的單核球/巨噬細胞功能測試 …………….……………………………....... 37
第五章 討論 ……………………………………….……………………………...……… 42
第六章 參考資料 ………………………….………….…………………..........……...... 46

圖表目錄

圖一 周邊血液單核細胞純化後結果 ……………………………….………...…..… 53
圖二 qPCR檢測不同細胞中GLA基因表現量 …...………………….……………. 54
圖三 細胞內GLA蛋白質免疫螢光染色結果 ……………………….….…….…….. 55
圖四 流式細胞儀檢測不同細胞中GLA基因表現量 ……………….…………...…. 56
圖五 法布瑞氏症小鼠的配種方法 …………………………………….….……….... 57
圖六 小鼠脾臟免疫細胞組成分群 ……………………………………..…………… 58
圖七 6-8 週老鼠的免疫細胞群變化狀態 …………………………….….…..…..… 59
圖八 12-15 週老鼠的免疫細胞群變化狀態 ……………………….……….....…… 60
圖九 20 週老鼠的免疫細胞群變化狀態 …………………………….…….……..... 61
圖十 17-34週老鼠的免疫細胞群變化狀態 ……………………………..…………. 62
圖十一 THP-1細胞中GLA基因表現量 ………………………………….…….……... 63
圖十二 Crispr KO GLA-THP-1細胞中GLA蛋白表現量 ……………….………........ 64
圖十三 經藥物刺激後的人類單核球細胞上清液中IL-1β表現量 ……...…..……….. 65
圖十四 藥物刺激後人類單核球細胞表現的IL-1β變化狀態 ………………….……... 66
圖十五 藥物刺激後健康受試者個體的IL-1β變化狀態 ………………....…..……..... 67
圖十六 藥物刺激後典型法布瑞氏症個體的IL-1β變化狀態 …………….………...... 68
圖十七 藥物刺激後IVS4個體的IL-1β變化狀態 ………………...…..…….……….... 69
圖十八 老鼠骨髓趨細胞分化為巨噬細胞的成效 ……………………..…….…...…… 70
圖十九 老鼠骨髓趨化巨噬細胞的Lysotracker染色 …………………….……….….. 71
圖二十 老鼠骨髓趨化巨噬細胞的CD77染色 …………………………....……….…. 72
圖二十一 比較不同基因型老鼠骨髓趨化巨噬細胞的染色結果 …………..…....……... 73
圖二十二 羊紅血球刺激後,老鼠骨髓趨化巨噬細胞的Lysotracker染色…….……..…. 74
圖二十三 羊紅血球刺激後,老鼠骨髓趨化巨噬細胞的CD77染色 ………….……...…. 75
圖二十四 骨髓趨化巨噬細胞經羊紅血球刺激後的染色結果 ……………..…………… 76
圖二十五 老鼠骨髓趨化巨噬細胞的吞噬能力檢測結果 ………………..…….….......... 77
圖二十六 比較老鼠骨髓趨化巨噬細胞的螢光訊號變化狀態 ………..…….……...…… 78
圖二十七 比較老鼠骨髓趨化巨噬細胞內的螢光強度 ……………..…….………...…… 79
表一 周邊血液單核細胞純化後結果 ………………………..…….…………...…… 53
表二 法布瑞氏症病人資料列表 ……………………………..…….………...……… 80

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