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    題名: Terbinafine誘發人類表皮癌細胞A431週期停滯及凋亡之分子機制研究
    Studies on the Molecular Mechanisms of Terbinafine-induced G0/G1 Cell Cycle Arrest and Apoptosis in Human Epidermoid Carcinoma A431 Cells
    作者: 陳元孝
    Yuan-Shiau Chen
    貢獻者: 醫學檢驗暨生物技術學研究所
    關鍵詞: 人類表皮癌
    細胞凋亡
    細胞週期
    Human Epidermoid Carcinoma
    Apoptosis
    Cell Cycle
    日期: 2003
    上傳時間: 2009-09-10 17:21:38 (UTC+8)
    摘要: Terbinafine，以下簡稱TB，是臨床上時常被使用的口服抗黴菌藥物，由於病人服用後藥物會集中在皮膚，於角質層及皮脂達到一相當高之濃度並維持長時期，因此本研究目的在於探討TB對人類皮膚癌是否具有治療的潛力以及作用機制。在生長曲線的實驗我發現，相較於Hs695T和RPMI 7951這兩株人類黑色素瘤細胞，TB抑制人類表皮癌細胞A431的生長效果較為明顯。經由流式細胞儀和DNA電泳分析，我也發現120 M劑量的TB處理24和48小時會造成A431細胞週期G0/G1、subG1比例的上升和DNA裂片的增加，顯示TB引發A431細胞死亡是經由G0/G1細胞週期停滯導致的計畫性死亡而非壞死。在分析蛋白量變化我同樣也看到120 M劑量的TB處理24和48小時促使A431細胞內Rb表現量增加，Bcl-2表現量下降，caspase 3、caspase 8、caspase 9等蛋白活化。利用免疫螢光染色在雷射共軛焦點螢光顯微鏡下觀察，我發現cytochrome c和AIF ( apoptosis inducing factor )在經過TB刺激24小時後會從粒線體分別轉位到細胞質以及細胞核中，也觀察到p-JNK和Bax分別在TB處理1和6小時轉位到粒線體， Bcl-2和14-3-3σ蛋白位置在6小時內沒有變化。以JC-1染色發現TB會造成粒線體膜電位在1小時下降。在DCFH-DA染色也發現TB處理5分鐘細胞內ROS量也有增加。綜合我上述發現的結果，我認為TB引發人類皮膚癌細胞A431週期停滯及凋亡，具有治療皮膚癌的潛力。
    TB (Terbinafine) is a clinical antifungal drug. It accumulates in skin tissue soon after taken by patient and then reach a high level for a long period of time. Therefore I exam the possibilities of TB being an anti-skin cancer medicine and forward study the molecular mechanisms. In the growth curve experiment, it is found that TB has caused more significant growth inhibition in A431 human epidermoid carcinoma cells than in Hs695T and RPMI 7951 human melanoma cells. According to the data of flow cytometry and DNA electrophoresis suggesting that TB induces G0/G1 cell cycle arrest and apoptosis instead of necrosis in A431 cells. It is also observed an increase in Rb protein level, decrease in Bcl-2 protein level and caspase 3, 8, 9 cleavage activation in 24hr and 48 hr. Using immunostaining under laser confocal microscopy observation it is also found that cytochrome c and AIF (apoptosis inducing factor) translocate from mitochondria intermembrane space to cytosol and nuclei respectively in 24hr, and p-JNK and Bax also translocate from cytosol to mitochondria in 1 and 6hr respectively. There is no sign showing Bcl-2 and 14-3-3σ protein translocation in 6hr. TB also causes a loss of mitochondria membrane potential in 1hr by JC-1 staining and a ROS generation in 5 min by DCFH-DA staining. To sum up, TB has agreat potential treatment in clinical skin cancer therapy.
    資料類型: thesis
    顯示於類別:[醫學檢驗暨生物技術學系所] 博碩士論文

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