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系統識別號 U0026-1301201208152800
論文名稱(中文) Fluconazole在離體下對細胞色素3A4表現層次抑制作用的機制探討
論文名稱(英文) Inhibition of CYP3A4 expression level in vitro by fluconazole
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 100
學期 1
出版年 101
研究生(中文) 王國安
研究生(英文) Kuo-An Wang
學號 s26981018
學位類別 碩士
語文別 中文
論文頁數 49頁
口試委員 指導教授-黃金鼎
指導教授-賴明亮
口試委員-周辰熹
中文關鍵字 細胞色素  酵素活性 
英文關鍵字 fluconazole  CYP3A4  in vitro 
學科別分類
中文摘要 Cytochrome P450是參與外來物質氧化代謝重要的酵素,其中的CYP3A4可以代謝將近50%的臨床用藥。Azole類抗黴菌藥物被報導與臨床上其它經由CYP3A4所代謝的藥物共同服用時,會抑制酵素活性,產生藥物交互作用,使得藥物血中濃度上升,對人體造成毒性。Ketoconazole為最常見的azole類抗黴菌藥物,不只會抑制CYP3A4的酵素活性,也會破壞CYP3A4基因上游細胞核接受體PXR,造成酵素轉錄、轉譯層次降低;fluconazole,屬於帶有含triazole官能基的azole類抗黴菌藥物,被認為對CYP3A4酵素活性抑制的效果較弱。然而,針對臨床上fluconazole所使用的濃度,對於CYP3A4在轉錄、轉譯方面上的影響並沒有被報導,因此想觀察fluconazole是否會對CYP3A4的mRNA或蛋白質造成抑制進行探討。
利用HepG2(人類肝癌細胞株)、Caco-2(結腸線癌細胞株)及LS174T (大腸結腸癌細胞株)進行實驗。首先以acid phosphatase (ACP) assay確認加入臨床上所使用不同濃度的fluconazole,細胞存活率仍維持在80%以上。已知rifampin會透過PXR誘導CYP3A4,透過P450-GLOTM assay發現,在rifampin誘導的情況下,fluconazole對CYP3A4酵素活性的抑制能力隨著濃度的上升而增強,濃度愈高則酵素活性愈低。另外以RT-PCR觀察CYP3A4轉錄層次的表現,發現rifampin存在時,fluconazole並不會減低CYP3A4 mRNA的表現量;同時也利用西方點墨法觀察CYP3A4轉譯的情況,其結果與RT-PCR相似,即fluconazole濃度的增加並不會改變蛋白質表現量。
從上述實驗結果發現,fluconazole對CYP3A4的抑制作用並非在轉錄、轉譯層次,而是酵素活性方面。推測fluconazole可能藉由改變CYP3A4蛋白質結構,使受質無法與酵素催化區結合進行代謝。
英文摘要 Cytochrome P450 proteins are kinds of oxidative enzymes which play important roles in the xenobiotics metabolism. CYP3A4 is responsible for the oxidation of about 50% clinical used drugs. Ketoconazole, a clinically used antifungal drug, inhibits not only the activity of CYP3A4 on the enzymatic level, but also the transcription of CYP3A4. Fluconazole, another antifungal drug, is the azole compound with triazole functional group and inhibits CYP3A4 activity in in vivo studies. However, the effect of fluconazole in clinical concentration on CYP3A4 expression has not been established.
In order to confirm whether fluconazole would affect CYP3A4 enzyme activity, we used P450-GloTM CYP3A4 assay to measure CYP3A4 activity in HepG2, Caco-2 and LS174T cell lines. Before the further study about inhibition of CYP3A4 by fluconazole in clinical concentration, cell viability was checked to be above 80%. Our results show that fluconazole decreased enzyme activity in a dose-dependent manner. We used the Western Blot and RT-PCR to detect CYP3A4 expression and found that fluconazole did not decrease both mRNA and protein expression.
Fluconazole appears to act via noncompetitive inhibition by rapid, reversible binding of the inhibitors or its metabolites of CYP3A4. The study suggests that fluconazole does not inhibit CYP3A4 through transcription or translation level.
論文目次 目錄
中文摘要 I
Abstract III
誌謝 IV
目錄 VI
圖目錄 IX
縮寫檢索表 X
壹.緒論 1
一. 前言 1
二. 細胞色素P450(Cytochrome P450) 2
三. 細胞色素3A4 (Cytochrome P450,family 3,subfamily A,polypeptide 4;CYP3A4) 3
四. Azole類抗黴菌藥物 4
五. Fluconazole 5
六. CYP3A4抑制機制 6
七. 研究動機 8
貳.實驗材料 9
一. 化學藥品 9
二. 試劑組 12
三. 抗體 12
四. 細胞培養相關材料 13
五. 實驗儀器 14
六. 其它材料 15
参.實驗方法 16
一. 細胞培養 16
二. 實驗藥品配製 17
三. 細胞存活率測定 17
四. CYP3A4酵素活性測定 18
五. CYP3A4 mRNA 表現量分析 18
五.1全量RNA萃取 18
五.2反轉錄反應(Reverse transcription, RT) 20
五.3 聚合酶連鎖反應(Polymerase chain reaction, PCR) 21
六. CYP3A4蛋白質表現量分析 22
六.1 蛋白質樣本製備與蛋白質濃度測定 22
六.2西方點墨法(Western blot) 23
肆.實驗結果 27
一. Azole類抗黴菌藥物對CYP3A4酵素活性的抑制現象 27
二. Fluconazole對不同細胞產生的細胞毒性 28
三. Fluconazole對CYP3A4酵素活性的抑制現象 28
四. Fluconazole不會抑制CYP3A4 mRNA及蛋白質表現量 28
伍.結論與討論 31
參考文獻 35
自述 49

Figure 1. Fluconazole structure. 41
Figure 2. Azole compounds affect rifampin-mediated induction of CYP3A4 enzyme activity. 42
Figure 3. Cell survival of HepG2, Caco-2 or LS174T cells for rifampin, fluconazole and treatment. 43
Figure 4. CYP3A4 enzyme activity was affected by fluconazole. 44
Figure 5. CYP3A4 mRNA and protein expression were not inhibited by fluconazole on HepG2 cells. 45
Figure 6. CYP3A4 mRNA and protein expression were not inhibited by fluconazole on LS174T cells. 46
Figure 7. CYP3A4 mRNA and protein expression were not inhibited by fluconazole on Caco-2 cells. 47
Figure 8. The mechanism of non-competitive inhibition. 48
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