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研究生:呂孟鎧
研究生(外文):Meng-Kai Lu
論文名稱:探討斑馬魚的Atox1在細胞內抗氧化反應所扮演的角色
論文名稱(外文):Characterization of Zebrafish Atox1 and its role on anti-oxidative stress
指導教授:蔡淦仁
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:醫學檢驗暨生物技術學系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:40
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銅離子是動物體內必要的微量元素,它可以作為不同酵素的輔因子提供酵素活性。在低濃度狀況下,銅離子可以幫助細胞正常生長,但是當銅離子過度堆積時會造成毒性,進而產生疾病甚至導致癌症。Atox1是一個金屬離子伴護蛋白,在銅離子恆定中扮演著重要的角色。它可將銅離子送往ATP7A及ATP7B以利銅離子的運輸。近期研究也發現,Atox1還可以成為轉錄因子(transcription factor, TF)調控SOD-3的基因轉錄。但是對於Atox1直接參與抗氧化的機制尚不明確,所以本篇研究中,我利用斑馬魚來研究Atox1蛋白在斑馬魚發育中抗氧化機制所扮演的角色。利用顯微注射Atox1 Morpholino的方式,觀察斑馬魚在缺乏Atox1蛋白的狀況下對於其抗氧化能力的影響。當Atox1蛋白表現量下降時,由DCFH-DA染色可以檢測出斑馬魚體內堆積較多的活性氧化物。此外Acridine Orange染色也可以發現,斑馬魚胚胎發育早期若缺乏Atox1蛋白,斑馬魚體內會引發較多的凋亡細胞。由此可知,缺乏Atox1蛋白可能使體內活性氧化物堆積,進而促使細胞凋亡的發生。因此,Atox1蛋白在斑馬魚發育中扮演著重要的抗氧化角色。

Copper is an essential element in living organisms as it is an important cofactor for many enzymes. At low concentration, copper is required for normal cell growth, whereas it is extremely toxic at a higher concentration. Atox1, a copper metallochaperone, plays an important role in the copper homeostasis. Atox1 transfers copper to the copper exporting P-type ATPases ATP7A and ATP7B. Recently, a novel function for Atox1 as a transcription factor (TF) for SOD-3 was reported. However, the relationship between Atox1 and antioxidant activity is not clear. In this study, zebrafish was used as an animal model to investigate the role of Atox1 in cellular redox reaction during zebrafish embryonic development. The Atox1-defieicnt zebrafish was established by using Atox1-MO to knockdown Atox1 expression. Upon Atox1 knockdown, increased reactive oxygen species (ROS) was measured in zebrafish embryo by DCFH-DA. In addition, Acridine Orange staining showed that Atox1 knockdown induced cell death in zebrafish embryo. These results revealed that Atox1 deficiency enhances ROS accumulation and apoptosis. Thus, an anti-oxidative and an anti-apoptotic role of Atox during zenrafish embryonic development is established.

目錄
中文摘要 i
英文摘要 ii
第一章、緒論 1
第一節、銅離子 1
第二節、銅離子所引發的疾病 2
第三節、銅離子所造成的氧化損傷 3
第四節、活性氧化物 4
第五節、細胞內的抗氧化機制 5
(一)超氧化物歧化酶 5
(二)過氧化氫酶 6
(三)榖胱甘肽 6
第六節、銅離子伴護蛋白 Atox1 7
第七節、研究動機 9
第二章、實驗材料與方法 10
一、斑馬魚養殖. 10
二、Atox1 蛋白對 ABTS 自由基清除能力試驗 10
三、反義寡核苷酸與顯微注射(Microinjection) 10
四、Total RNA 萃取與反轉錄作用(Reverse transcription) 11
五、即時聚合酶連鎖反應(Real-time PCR) 12
六、斑馬魚胚胎內自由基染色(DCFH-DA staining) 12
七、斑馬魚胚胎細胞凋亡染色(Acridine Orange staining) 13
八、流式細胞儀(Flow Cytometry) 13
九、蛋白萃取 14
十、西方墨點法(Western Blot) 14
第三章、實驗結果 15
一、各物種間 Atox1 蛋白的序列比對 15
二、斑馬魚 Atox1 蛋白在體外實驗可清除 ABTS 自由基 15
三、Atox1 蛋白在斑馬魚體內有抗氧化能力 16
四、活性氧化物會可能會活化 Atox1 基因表現 19
五、斑馬魚缺乏 Atox1 蛋白會引發細胞凋亡 20
六、利用流式細胞儀定量 DCFH-DA 與 Acridine Orange 染色結果 20
七、斑馬魚 Atox1 蛋白缺乏可能影響細胞凋亡內在路徑 21
第四章、討論 22
第五章、圖表說明 25
Fig. 1 各物種間 Atox1 蛋白序列比對 25
Fig. 2 Atox1 蛋白對 ABTS 自由基清除能力試驗 26
Fig. 3 不同濃度 Atox1 MO 對於斑馬魚存活率的影響 27
Fig. 4 斑馬魚 DCFH-DA 染色結果 28
Fig. 5 RNA rescue 後斑馬魚體內 ROS 累積情形 29
Fig. 6 斑馬魚注射 Atox1 MO 後 12hpf 基因表現情形 31
Fig. 7 斑馬魚注射 MO 後體內細胞凋亡情形 32
Fig. 8 DCFH-DA 及 Acridine Orange 染色利用流式細胞儀定量結果 33
Fig. 9 細胞凋亡內在路徑 BCL2 蛋白 Westeren Blot 結果圖 34
參考資料 35
附錄 39

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