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研究生:詹鈺珮
研究生(外文):Yu Pei Chan
論文名稱:蜜蜂磁鐵礦化機轉研究
論文名稱(外文):The mechanism of Magnetite Biomineralization in Honeybees (Apis melliera)
指導教授:徐錦源徐錦源引用關係
指導教授(外文):C. Y. Hsu
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:110
中文關鍵詞:蜜蜂磁鐵礦化鐵囊胞鐵蛋白
外文關鍵詞:HoneybeesMagnetite Biomineralizationiron deposition vesiclesferritin
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蜜蜂能在細胞內沉積磁鐵礦物,但是磁鐵沉積機轉至今不明。本研究中,我們將純化鐵顆粒(IGs)及鐵囊胞(IDVs)蛋白,利用蛋白質體技術預測其可能的生物功能,培養這些蛋白的抗體,利用冷凍電顯免疫技術及免疫螢光法確認蛋白在鐵顆粒(IGs)及鐵囊胞(IDVs)中分佈的位置。目前結果顯示,在鐵顆粒(IGs)及鐵囊胞(IDVs)中主要蛋白分別有actin、ferritin 2、myosin與ATP synthase。經共厄焦顯微鏡及穿透式電子顯微鏡分析確認,證實actin、ferritin 2、myosin與ATP synthase存在於鐵囊胞中。此外,利用免疫沉澱法發現actin-myosin-ferritin 2會互相聯結。我們推測,ferritin 2可能參與形成7.5 nm鐵顆粒,並藉由actin與myosin協助運送7.5 nm鐵顆粒進入到鐵囊胞中沉積,而ATP synthase則提供運送時所需要的能量。
Honeybees can intracellularly deposit iron minerals into magnetite. However, the mechanism of iron biomineralization is unclear. In this study, the proteins in/on iron granules (IGs) and iron deposition vesicles (IDVs) were purified, incubated into antibodies, labeled the location in/on IGs and IDVs by biochemical and microscopic techniques. The results showed that the major proteins in/on IGs and IDVs were actin, myosin, ferritin 2, and ATP synthase. They had been demonstrated to exist in/on IGs and IDVs by using confocal microscopic and transmission electron microscopic assays. In addition, actin was associated with ferritin 2 by immunoprecipitation analysis. Collectively, ferritin 2 possibly participated the formation of 7.5 nm particles, actin and myosin served as the transporter of 7.5 nm particle for iron deposition, and ATP synthase supplied energy for transportation.
目 錄
指導教授推薦書…………………………………………………………...
口試委員會審定書………………………………………………………...
授權書…………………………………………………………………...iii
誌謝……………………………………………………………………...iv
中文摘要…………………………………………………………………v
英文摘要………………………………………………………………...vi
目錄……………………………………………………………………..vii
圖表目錄………………………………………………………………...ix
第一章 緒論……………………………………………………………1
1-1 生物礦化的發展………………………………………………..2
1-2 生物礦化的作用機制…………………………………………..3
1-3 鐵元素…………………………………………………………..5
1-4 生物性鐵質沉積………………………………………………..7
1-5 磁鐵礦之生物礦化機制……………………………. …………9
1-6 最佳模式生物:蜜蜂(Apis mellifera)………..........12
研究動機………………………………………………………………...18
研究目的………………………………………………………………...19
研究設計………………………………………………………………...20
第二章 材料與方法…………………………………………………….21
第三章 結果…………………………………………………………….41
3-1 純化鐵顆粒(IGs)與鐵囊胞(IDVs)蛋白..…………………….42
3-2 分析鐵顆粒(IGs)與鐵囊胞(IDVs)蛋白……………………...42
3-3 專一性抗體製備與鑑定…………….………………………..43
3-4 抗體在營養細胞(trophocytes)的分佈………………………..43
3-5 專一性抗體在鐵囊胞(IDVs)的分佈…………………………44
3-6 Ferritin 2共同分佈表現蛋白探討……………………………47
3-7 Ferritin 2共同交互作用蛋白探討……………………………47
第四章 討論……………………………………………………………49
4-1 建立鐵顆粒(IGs)與鐵囊胞(IDVs)純化技術………………...50
4-2 參與磁鐵沉積相關蛋白分析………………………………...51
4-3 鐵囊胞(IDVs)中蛋白質的分佈與共同聯結表現……………55
第五章 結論……………………………………………………………57
第六章 圖說……………………………………………………………59
參考文獻…………………………………………….………………….86



表目錄
表2-1 質譜分析法(MALDI TOF/TOF Mass Spectrometer)分析鐵顆粒(IGs)與鐵囊胞(IDVs)中蛋白…………………………………..66
表2-2 質譜分析Actin、Myosin、Ferritin 2與ATP synthase的結果....67















圖目錄
圖1-1 趨磁細菌TEM圖………………………………………………60
圖1-2 蜜蜂腹部示意圖………………………………………………...60
圖1-3 蜜蜂腹部之腹板節片與細胞分佈之光學顯微鏡圖…………...61
圖1-4 蜜蜂營養細胞內鐵顆粒分佈之TEM圖……………………….61圖1-5 鐵沉積囊胞(IDVs)發育過程之TEM圖……………………….62
圖1-6 鐵沉積囊胞(IDVs)的純化技術示意圖………………………...63
圖1-7 純化自營養細胞的鐵顆粒(IGs)之TEM圖……………………64
圖1-8 純化自營養細胞的鐵顆粒之元素分析圖……………………...64
圖1-9 初步磁鐵沉積機轉假說………………………………………...65
圖2-1 純化鐵顆粒(IGs)技術示意圖…………………………………..68
圖2-2 純化鐵沉積囊胞(IDVs)技術示意圖…………………………...69圖2-3 純化自營養細胞的鐵顆粒(IGs)與鐵囊胞(IDVs)之TEM圖….70
圖2-4 分離純化後鐵顆粒(IGs)與鐵囊胞(IDVs)的蛋白成份...............71
圖2-5 鑑定Actin、Myosin、Ferritin 2與ATP synthase在脂肪體細胞(fat body)中的辨識度..................................................................72
圖2-6 Actin、Myosin、Ferritin 2與ATP synthase在營養細胞及鐵囊胞中的分佈圖………………………………………………………73
圖2-7 Actin在鐵囊胞中分佈的TEM圖與分佈統計圖...............……74
圖2-8 Myosin在鐵囊胞中分佈的TEM圖與分佈統計圖……………76
圖2-9 Ferritin 2在鐵囊胞中分佈的TEM圖與分佈統計圖.................78圖2-10 ATP synthase在鐵囊胞中分佈的TEM圖與分佈統計圖…….80
圖2-11 Ferritin 2與Acrin在營養細胞及鐵囊胞中分佈的共軛焦顯微鏡圖..................................................................................................82
圖2-12 Ferritin 2與ATP synthase在營養細胞及鐵囊胞中分佈的共軛焦顯微鏡圖……………………………………………………..83
圖2-13 Ferritin 2-Actin與Ferritin 2-Myosin免疫沉澱分析圖..………84
圖2-14 磁鐵沉積機轉假說模式圖…………………………………….85
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