胞外域蛋白 (ectodomain) 脫落是一種透過蛋白酶將細胞表面蛋白切除並釋放於外部環境的水解形式，此機制已被發現能調節包括細胞黏附、生長因子之訊號傳遞、發炎反應及細胞的存活等多種細胞行為。現今主要是透過免疫學方法分析分泌於培養液中的胞外域蛋白，然而其應用受限仍大。在本篇論文中，我們以親和性蛋白標記螢光探針作為研究胞外域蛋白的新策略。透過此方法，我們首次揭示了腫瘤缺氧指標之一的碳酸酐酶XII (CAXII) 其胞外域的脫落。相較於傳統的免疫學偵測，這類蛋白標記方法除了能夠測量培養液中的胞外域蛋白外，更能藉由細胞影像追蹤螢光訊號以及定量分析細胞表面剩餘的蛋白，提供了更加詳細的胞外域蛋白脫落以及蛋白通量的數據。我們相信此蛋白標記螢光探針的策略能改善免疫學方法的不足，在胞外域蛋白脫落的研究上成為一重要的工具。

Ectodomain shedding is a form of limited proteolysis where proteases cleave cell surface proteins resulting in the release of the extracellular domain. Cells use this mechanism to regulate a wide variety of cellular events, including growth factor signaling, cell adhesion, inflammatory responses and cell survival. Typically, immunological methods are employed for the detection of ectodomain secreted to the cultured medium. In this thesis, we described a new strategy by using affinity-based protein labeling fluorescent probe to study ectodomain shedding. With this approach, we have revealed in the first time the ectodomain shedding of cell surface carbonic anhydrase XII (CAXII) which is an important biomarker for tumor hypoxia. Several important shedding parameters were determined, including basal and stimulated shedding rates, protein half-life on the cell surface, and metalloprotease responsible for the ectodomain shedding. As compared with the immunological detection methods, the protein labeling approach not only enables detection of ectodomain released to the culture medium, but also real-time living cell tracking and quantitative analysis of remnant proteins on the cell surface, providing a more accurate and detail analysis of the ectodomain shedding as well as protein turnover. We believe that this protein labeling fluorescent probe approach can be a versatile tool to complement classical immunological methods to study ectodomain shedding of membrane proteins.

摘要
Abstract
謝誌
目錄
第一章、緒論-------------------------------1
1-1 細胞訊息傳遞 (Signal Transduction)-----1
1-2 訊息傳遞的機制-------------------------2
1-2-1 磷酸化 (Phosphorylation)------------2
1-2-2 糖基化 (Glycosylation)--------------4
1-2-3 蛋白水解 (Proteolysis)--------------6
1-3 胞外域蛋白脫落 (Ectodomain Shedding)---8
第二章、文獻回顧---------------------------12
2-1 免疫學偵測法 (Immunological Detection Methods)---12
2-1-1 免疫沉澱法 (Immunoprecipitation)---------------12
2-1-2 西方墨點法 (Western Blotting)------------------14
2-1-3 免疫學方法的缺點-------------------------------16
2-2 化學標記方法-------------------------------------17
2-2-1 放射性同位素標記法 (Radioisotopic Labeling Method)---17
2-2-2 生物素標記法 (Biotin Functionalized Method)----------18
2-2-3 非專一性化學標記方法的缺點----------------------------19
2-3 親和性螢光標記-----------------------------------------20
第三章、碳酸酐酶及其標記探針設計----------------------------22
3-1 碳酸酐酶 (Carbonic Anhydrases, CAs)-------------------22
3-2 螢光探針設計------------------------------------------23
第四章、實驗結果與討論-------------------------------------26
4-1 探針之基本性質探討------------------------------------26
4-1-1 探針1與重組蛋白hCAII之反應性測試---------------------26
4-1-2 探針1與重組蛋白hCAII之反應動力學---------------------27
4-1-3 探針1對重組蛋白hCAII之標記效率-----------------------28
4-1-4 探針1於複雜環境下之選擇性測試------------------------30
4-1-5 探針1之抗酯酶水解測試-------------------------------31
4-1-6 探針1之穩定性測試----------------------------------32
4-1-7 探針1之目標蛋白偵測極限-----------------------------34
4-2 探針1之細胞實驗--------------------------------------34
4-2-1 A549及MCF-7細胞影像之反應性實驗---------------------35
4-2-2 A549及MCF-7細胞裂解液之反應性實驗-------------------36
4-2-3 A549及MCF-7細胞影像之時程實驗 (Time-Course Experiments)---37
4-2-4 A549及MCF-7細胞裂解液之時程實驗---------------------------38
4-2-5 A549及MCF-7細胞裂解液與培養液結果之比較------40
4-2-6 A549及MCF-7細胞胞外域蛋白之活性測試----------41
4-2-7 A549及MCF-7細胞培養液之時程實驗-------------41
4-3 細胞之藥物實驗探討----------------------------42
4-3-1 金屬蛋白酶活化及抑制之藥物測試---------------42
4-3-2 A549及MCF-7細胞在PMA藥物活化下之時程實驗-----46
4-3-3 A549細胞於模擬缺氧環境下之時程實驗-----------49
第五章、總結--------------52
第六章、實驗部分----------53
6-1 實驗藥品與儀器--------53
6-2 蛋白質表達與純化------54
6-2-1 蛋白質表達---------54
6-2-2 蛋白質純化---------55
6-3 SDS-PAGE膠體電泳-----56
6-3-1 製膠配方-----------56
6-3-2 膠體電泳過程-------57
6-4 細胞培養及繼代流程----59
6-4-1 培養液與試劑-------59
6-4-2 細胞繼代培養-------59
6-5 細胞裂解液之SDS-PAGE實驗---------60
6-6 培養液之SDS-PAGE電泳分析實驗-----61
6-7 細胞影像實驗--------------------61
6-8 西方墨點法----------------------62
6-9 細胞存活率分析 (MTT試驗)--------63
第七章、參考資料--------------------65
附錄----------------72

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