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研究生： 嚴淑芳
Yen, Shu-Fang
論文名稱： 具活性之雌二醇衍生化及其與雌激素受體的鍵結分析之應用
The derivative of active estrodiol and its application of binding with estrogen receptor
指導教授： 陳淑慧
Chen, Shu-Hui
學位類別： 碩士
Master
系所名稱： 理學院 - 化學系碩士在職專班
Department of Chemistry (on the job class)
論文出版年： 2006
畢業學年度： 94
語文別： 中文
論文頁數： 89
中文關鍵詞： 雌二醇 、雌激素
外文關鍵詞： estrogen
相關次數： 點閱：86  下載：1 
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    • 雌激素影響著人體的生長發展以及細胞再生，許多雌激素所引發的效應都是透過與雌激素受體鍵結而發生。近幾年微流體晶片已被逐漸使用於分離科學上，雖然有許多優點，如分離時間短、樣品用量少和可拋式設計，但仍然有許多限制例如偵測靈敏度。如同毛細管電泳一樣，微流體晶片電泳的靈敏度仍受限於其通過的光學路徑非常短，所以不容易偵測低濃度的樣品，然而這種限制可藉由結合線上濃縮技巧的電泳分離得到改善。這裡我們將此技術應用於分析雌激素，將微包電動力毛細管層析結合線上濃縮用於普遍用在檢測鍵結效率的高鹽類溶液，藉由改變緩衝溶液中的組成去探討雌激素的濃縮倍率可達到200至300倍。
    為了發展晶片上螢光含量的偵測方法有系統的建立七個步驟的螢光標示合成步驟。最後產物利用高液相層析儀純化將其純度提高至98%以上。此產物Cy3-17β-estradiol (Cy3-E2)的結構也進一步利用NMR和MS做鑑定。我們將合成好的Cy3-E2應用於微流體晶片電泳的鍵結活性分析，結果顯示合成物質和雌激素受體具有鍵結活性。此外，在加入抗雌激素物質DES鍵結活性將變弱。這些結果證實了產物的合成成功，也證明了所提出含量具有可用性。

    Estrogen influences the growth, development, behavior and regulation of reproductive tissues in all vertebrates. Many of the effects of estrogens are mediated through its binding to the estrogen receptor (ER). The use of microchips in separation sciences has gained great interest in recent years. Despite that many advantages such as the short separation time, low sample amount and disposable use, there are still limitations such as the low detection sensitivity associated with this technique. Similar to capillary electrophoresis, the sensitivity of microchip electrophoresis is limited by the short optical path length of the microchannel. It is difficult to detect analytes with low concentrations. This limitation, however, can be improved by coupling electrophoretic separation with on-line concentration. Here we apply micellar electrokinetic chromatography with high salt on-line concentration to develop an assay for analyzing estrogens and estrogen binding. The enhancement factor for estrogens was investigated by varying the buffer composition for the analyte and up to 200 – 300 folds of enhancement were gained by this method.
    To develop an on-chip fluorescence assay, a fluorescence labeling procedure that involves seven synthesis steps was systematically optimized to obtain the highest purity of the product. The final product was purified by HPLC and the purity was estimated to be around 98%. The structure of Cy3-17β-estradiol (Cy3-E2), was further confirmed by NMR and MS. We apply the synthesized Cy3-E2 for binding activity experiment on microchip electrophoresis and the results indicate that the synthesized compound could bind to activated estrogen receptor. Moreover, as expected, the binding was weakened by a known anti-estrogenic compound, DES. These results confirm the success of the synthesis and also prove the usefulness of the proposed assay.

    第1 章序論 1-1 前言…………………………………………………1 1-2 類固醇………………………………………………2 1-3 雌激素與雌激素接受器……………………………3 第2 章晶片上電場驅動流體之原理 2-1 電泳原理……………………………………………4 2-1-1 界面電位及電滲流………………………………4 2-1-2 晶片電泳…………………………………………6 2-2 電泳分離模式………………………………………6 2-2-1 微胞電動力毛細管層析法………………………7 2-2-2 MEKC 分離機制……………………………………8 2-3 線上濃縮機制………………………………………10 2-3-1 堆疊………………………………………………10 2-3-2 清掃堆積…………………………………………12 2-3-3 高鹽類濃度樣品堆積效應………………………14 2-4 結合壓力推動及電壓驅動進樣模式………………15 2-5 微流體晶片電壓結合壓力驅動模式………………16 2-5-1 微流體晶片設計…………………………………16 2-5-2 晶片式電泳儀及晶片樣式………………………17 2-5-3 微晶片管道前處理………………………………19 2-5-4 藥品與試劑………………………………………20 2-5-5 緩衝溶液及樣品溶液配製………………………20 2-5-6 微晶片電泳進樣及分離條件……………………21 2-6 結果與討論…………………………………………22 第3 章雌二醇衍生物合成 3-1 研究動機與目的……………………………………23 3-2 器材與藥品…………………………………………24 3-2-1 器材………………………………………………24 3-2-2 藥品………………………………………………25 3-3 雌激素衍生化………………………………………26 3-4 螢光染料標示………………………………………30 3-4-1 標示步驟…………………………………………31 3-4-2 產物鑑定…………………………………………32 3-4-3 濃度定量…………………………………………32 第4 章線上濃縮及鍵結效率的分析 4-1 濃縮效率之探討……………………………………33 4-1-1 藥品………………………………………………34 4-1-2 緩衝溶液及樣品溶液配製………………………34 4-1-3 微晶片電泳進樣及分離條件……………………36 4-1-4 結果與討論………………………………………36 4-2 線上偵測Cy3-E2 和ER 之鍵結錯合物……………38 4-2-1 實驗目的…………………………………………38 4-2-2 親合性毛細管電泳原理…………………………38 4-2-3 實驗架構…………………………………………39 4-2-4 分析物成分………………………………………40 4-2-5 實驗操作條件……………………………………41 4-2-6 結果與討論………………………………………42 4-2-6-1 Cy3-E2 鍵結活性……………………………42 4-2-6-2 類固醇相似藥物對鍵結錯合物之競爭性反應43 第5 章結論與未來展望 5-1 結論…………………………………………………44 5-2 未來展望……………………………………………45 參考文獻…………………………………………………46

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