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研究生:江佩樺
研究生(外文):Pei-HuaChiang
論文名稱:利用串聯式質譜儀定量分析MCF-7乳癌細胞的雌激素代謝物
論文名稱(外文):Quantitative Analysis of Estrogen Metabolites in MCF-7 breast cancer cells by Liquid Chromatography-Tandem Mass Spectrometry
指導教授:陳淑慧陳淑慧引用關係
指導教授(外文):Shu-Hui Chen
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系碩博士班
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:60
中文關鍵詞:液相層析儀串聯式質譜儀雌激素雌激素代謝物乳癌細胞
外文關鍵詞:LC-MS/MSestrogenestrogen metabolitesMCF-7 breast cancer cell
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雌激素與雌激素代謝物經由許多研究報告指出,這類小分子確實跟乳癌細胞的生長有關聯性;舉例來說,雌一醇(estrone,E1)或雌二醇(estradiol,E2)在它們的二號或四號位置接上氫氧基的這個反應過程中,會經過分子帶有自由電子的狀態,而此一狀態的分子會與去氧核醣核酸(DNA)上的鹼基結合,使得去氧核醣核酸發生點突變,進而造成細胞不正常增生變成腫瘤;然而不全然所有的雌激素和雌激素代謝物都是會造成乳癌的風險性提高,二號位置接上甲氧基的雌二醇就曾經有研究指出其藉由抑制血管新生的方式,達到抑止乳癌細胞增生的結果。
本研究利用液相層析儀加上串聯式質譜儀,並搭配將雌激素和雌激素代謝物以丹磺醯氯(Dansyl chloride)衍生化的方式(有較強的訊號發生在m/z為171時)提升偵測的靈敏度,定量分析MCF-7此一乳癌細胞當中的雌激素與雌激素代謝物。實驗中利用逆向層析管柱C18以三十五分鐘的梯度分離十三個雌激素與雌激素代謝物,分別是E1,E2,estriol (E3),2-hydroxyestrone (2-OHE1),4-hydroxyestrone (4-OHE1),2-hydroxyestradiol (2-OHE2) ,4-hydroxyestradiol (4-OHE2),2-methoxyestrone (2-MeOE1) ,4-methoxyestrone (4-MeOE1),2-methoxyestradiol (2-MeOE2),4-methoxyestradiol (4-MeOE2),16α-hydroxyestrone (16α-OHE1)以及內標準品ethinylestradiol (EE2)。使用乙酸乙酯以液液萃取的方式萃取培養液與全細胞萃取溶液當中的雌激素與雌激素代謝物,在培養液中平均回收率為105.5 %,在全萃取細胞溶液當中的平均回收率為97.2 % 。校正曲線的R2值皆大於0.99以上。在串聯式質譜儀的SRM模式下,培養液的偵測極限為0.02 pM─30.38 pM,全萃取細胞溶液的偵測極限為0.07 pM─74.95 pM。本研究方法用在與1×10-6 M雌二醇作用二十四小時的MCF-7乳癌細胞上,定量分析細胞內與細胞外的雌激素與雌激素代謝物;由實驗結果可以發現,在培養液當中大約存在730 nM的E2,而約有2.5 nM的E2進入細胞當中;除了16α-OHE1,在培養液當中有十種雌激素及雌激素代謝物(不包含E2)可被測得,其濃度範圍在0.2到220 nM之間;而在全細胞萃取溶液當中可測得約1 nM的E1和E2。

Estrogen metabolites are known to be involved in the growth of breast cancer cells. For example, 2-hydroxylation and 4-hydroxylation of 17?-estradiol (E2) or estrone (E1) will damage DNA and lead to mutations or cell proliferation. On the contrary, 2-methoxyestradiol inhibits the proliferation of breast cancer cells by disrupting microtubule function. This work develops a quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to analyze estrogen metabolites in MCF-7 cells through dansyl chloride derivatization, which gives a reporter ion m/z 171. The separation of 13 estrogen metabolites including E1, E2, estriol (E3), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 2-methoxyestrone (2-MeOE1), 4-methoxyestrone (4-MeOE1), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 16α-hydroxyestrone (16α-OHE1), and internal control ethinylestradiol (EE2) was completed by a 35-min LC gradient elution on a reversed phase C18 column. Estrogen metabolites were extracted by ethyl acetate (EA) from MCF-7 cell lysate and medium. The recovery yield of EA extraction reached 105.5 % for the medium and 97.2 % for the cell lysate. Calibration curves were constructed with R2 values greater than 0.99. The detection limits were determined be 0.02 pM─30.38 pM for the mdium and 0.07 pM─74.95 pM for the cell lysate under the selected reaction monitoring (SRM) mode. The developed method was then applied to investigate the metabolites of estrogens in extracellular as well as in cellular compartments of MCF-7 cells after a 24-hour treatment of 1×10-6 M of 17?-estradiol. Results indicate that about 730 nM E2 was present in the medium and about 2.5 nM E2 diffused into the cell. Except 16α-OHE1, all the rest 10 metabolites (exclude E2) ranging from 0.2 to 220 nM were detected in the medium. In addition, about 1 nM of E1 and E2 were detected in the cell lysate.
中文摘要 Ι
英文摘要 III
致謝 V
目錄 Ⅵ
表目錄 Ⅸ
圖目錄 Ⅹ
英文縮寫對照表 XII
第一章 : 序論 1
1.1: 代謝體學的發展 1
1.2: 不同的雌激素代謝物跟乳癌的關連性 2
1.3: 雌激素代謝物的檢測 8
1.4: 跟MCF-7乳癌細胞相關的研究 10
第二章 : 材料與實驗方法 14
2.1 : 實驗目的與實驗架構 14
2.2 : 儀器設備 18
2.3 : 藥品 18
2.4 : 細胞培養 19
2.5 : 細胞處理 19
2.6 : 全細胞萃取 20
2.7 : 培養液收集 20
2.8 : 酵素裂解 21
2.9 : 雌激素及雌激素代謝物萃取 21
2.10 : 衍生化方法 21
2.11 : 層析分離系統 22
2.12 : 質譜儀條件設定 22
2.13 : 雌激素及雌激素代謝物定量計算 23
2.14: 檢量線製備 23
2.15: 偵測極限 25
2.16: 前處理回收率 25
2.17: MRM Mode 離子對選擇 28
第三章 : 結果與討論 29
3.1 : 二次質譜圖 29
3.2 : 標準品LC-MS/MS圖譜 31
3.3 : 檢量線 35
3.4 : 偵測極限 41
3.5 : 前處理回收率 43
3.6 : 酵素裂解 46
3.7 : 定量結果 51
第四章 : 結論 57
參考文獻 : 58

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