由於MGMT的含量多寡與烷基化藥物的耐藥性相關，可作為化學治療的重要指標及有效的預後標記。而醫院每天須檢測大量的細胞樣品，故開發一種快速又簡單的分析方法，以檢測在不同類型的細胞中MGMT之含量是有其必要性。在本研究中，我們開發一種新穎的MGMT蛋白之螢光增益型探針。以分子轉子CCVJ為螢光基底，修飾上MGMT假性受質O6-BG。當與MGMT蛋白作用後，探針轉移至MGMT蛋白的活性位點。由於蛋白質為巨大結構之立體障礙影響，使分子轉子單鍵旋轉的自由度下降，產生170倍螢光增益效果。將探針與含MGMT表達之Hela S3、MCF-7及HEK293細胞株作用後，可觀察到明顯的螢光訊號，而缺乏MGMT表達之CHO細胞株則無螢光訊號產生。我們所設計的螢光探針容易合成，並具有標記速率快且免清洗之細胞成像等優勢。我們相信此螢光增益型探針的設計可延伸運用於偵測其他轉移酶或結合蛋白，並能成功地運用在活體細胞成像。

MGMT protein, which has been associated with resistance to antitumor alkylation drugs for many patients, is a very useful prognostic marker to provide a guide for therapeutic decisions. Considering the large number of cellular samples that have to be handled at the hospital every day, it is thus important to develop a rapid and simple analytical method to distinguish MGMT activity in different types of cells. In this study, we describe the first MGMT fluorescence activation probe for the rapid no-wash imaging of MGMT in living cells. The probe consists of a specific MGMT suicide pseudosubstrate, O6-benzylguanine and a fluorescent molecular rotor CCVJ. In the presence of MGMT, the enzyme transfers CCVJ to the protein active site where the crowded surrounding restricts the bond rotation of the fluorescent molecular rotor to trigger fluorescence activation of about 170-fold. With this probe, bright fluorescence was observed for MGMT-positive, Hela S3, MCF-7 and HEK293 cells, while MGMT-deficient CHO cells displayed no fluorescence. This fluorescence activation probe design can also be extended for the detection of other transferases and binding proteins for which there are still no effective methods to image them in living cells.

摘要 i
Abstract ii
誌謝 iii
著作列表 iv
目錄 v
第一章 緒論 1
1-1 蛋白質 1
1-2 偵測蛋白質之方法 3
1-2.1 質譜技術 3
1.2-2 西方點墨法 6
1-2.3 酵素連結免疫吸附分析 7
1-2.4 選擇性蛋白探針 8
第二章 文獻回顧 14
2-1 O6-methylguanine-DNA methyltransferase (MGMT) 14
2-2 MGMT之分析方法 15
2-2.1 甲基特異PCR (methylation-specific PCR, MSP) 15
2-2.2 免疫組織化學染色法(Immunohistochemistry, IHC) 19
第三章 螢光探針設計構想 27
3.1 螢光分子選擇 27
3.2 螢光探針設計構想 30
第四章 結果與討論 32
4-1 螢光探針性質測試 32
4-2 動力學分析及水解反應測試 39
4-3 細胞實驗 43
4-4 細胞中烷基化MGMT之降解 49
4-5即時追蹤細胞內烷基化MGMT之降解狀態 51
第五章 結論 53
第六章 實驗部分 54
6-1實驗藥品及器材 54
6-2 有機合成與光譜資料 56
6-3 動力學測試 61
6-4 蛋白質表現及純化 62
6-4.1 蛋白質表現 62
6-4.2 蛋白質純化 63
6-4.3 SDS-膠體電泳 63
6- 5 細胞培養及細胞影像實驗 65
6-5.1 培養基及試劑 65
6-5.2 細胞繼代培養 65
6-5.3 細胞轉染 66
6-5.3 細胞影像 66
6-5.4 細胞裂解液偵測 67
6-5.5 西方點墨法 68
參考文獻 69
附錄 73

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