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論文名稱 Title |
Isoobtusilactone A 在人類肝癌細胞株(Hep G2)所引起細胞凋亡之相關作用機制的探討 The mechanisms of isoobtusilactone A-induced apoptosis in human hepatoblastoma cell line (Hep G2) |
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系所名稱 Department |
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畢業學年期 Year, semester |
語文別 Language |
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學位類別 Degree |
頁數 Number of pages |
85 |
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研究生 Author |
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指導教授 Advisor |
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召集委員 Convenor |
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口試委員 Advisory Committee |
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口試日期 Date of Exam |
2006-07-23 |
繳交日期 Date of Submission |
2007-07-28 |
關鍵字 Keywords |
細胞凋亡、活性氧、Bax蛋白、粒線體膜電位、caspase 3蛋白、AIF蛋白 Isoobtusilactone A |
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統計 Statistics |
本論文已被瀏覽 5684 次,被下載 5157 次 The thesis/dissertation has been browsed 5684 times, has been downloaded 5157 times. |
中文摘要 |
使用天然植物所分離之物質進行化學預防(chemoprevention),已成為許多癌症預防的新策略,因此,本研究探討從蘭嶼肉桂(Cinnamomum kotoense)樹葉所分離出來的isoobtusilactone A化合物對Hep G2肝癌細胞株的毒殺作用及其導致細胞凋亡的可能機制。初步實驗結果發現隨著isoobtusilactone A濃度的增加,Hep G2細胞的sub-G1細胞群和小片段DNA斷裂(oligonucleosomal DNA fragmentation)也會跟著上升;進一步,在細胞蛋白的分析也證實了isoobtusilactone A會促使Bax蛋白從細胞質轉移到粒線體中,同時也將cytochrome c釋放至細胞質中,進而活化caspase 3 和PARP cleavage,最後導致Hep G2細胞凋亡;而且也發現isoobtusilactone A 會增加Hep G2細胞活性氧(ROS)的產生,誘導粒線體膜電位下降(mitochondrial transmembrane potential ),而當分別加入活性氧清除劑(NAC)和NADPH oxidase 抑制劑(DPI)時,發現其會抑制因藥物所誘發產生之ROS和細胞凋亡。另外,我們也發現當加入caspases抑制劑Z-VAD.fmk時,並不能完全的保護細胞走向細胞凋亡,意味著還有其它的細胞凋亡機制参與。Apoptosis-inducing factor (AIF) , 為調控caspase-independent apoptosis的蛋白,實驗結果發現,isoobtusilactone A 可促進AIF從粒線體轉移至細胞核內,並伴隨大片段DNA斷裂(large-scale DNA fragmentation)的產生。因此以AIF-siRNA短暫轉染(transient transfection) Hep G2 細胞,發現會抑制isoobtusilactone A誘發之大片段DNA斷裂,但卻不能抑制 isoobtusilactone A誘導小片段DNA斷裂;而同時以AIF-siRNA 和Z-VAD.fmk前處理Hep G2 細胞,則能同時抑制isoobtusilactone A誘導小片段和大片段DNA 斷裂。綜合上述,isoobtusilactone A 誘導Hep G2細胞凋亡路徑是經由ROS產生來破壞粒線體膜電位,進而同時誘發caspase-dependent and caspase-independent pathway來執行完成。 |
Abstract |
Chemoprevention using naturally occurring substances has now been considered a promising strategy for the prevention of cancer. In this study, the effects of isoobtusilactone A, a novel constituent isolated from the leaves of Cinnamomum kotoense, on the proliferation of human hepatoma Hep G2 cells and the underlying mechanisms in isoobtusilactone A-induced apoptosis are thoroughly evaluated. Under the experimental conditions adapted by this study, isoobtusilactone A was found to exhibit a concentration-dependent growth impediment (IC50 = 37.5 μM). The demise of the cells induced by isoobtusilactone A was apoptotic in nature, showing progressive sub-G1 fraction and DNA fragmentation when the concentration of the substrate was increased. Subcellular fractionation analysis further revealed that Bax translocation to mitochondria resulted in a rapid release of cytochrome c, followed by the activation of caspase 3 and PARP cleavage, and finally cell death. Isoobtusilactone A-treated cells also displayed transient increase of ROS during the earlier stage of the experiment, followed by the disruption of mitochondrial transmembrane potential ( m). The presence of a ROS scavenger (N-acetyl-L-cysteine,NAC) and an inhibitor of NADPH oxidase (diphenyleneiodonium chloride,DPI) blocked ROS production and the subsequent apoptotic cell death. Taken together, our data suggest that ROS generated through the activation of NADPH oxidase plays an essential role in apoptosis induced by isoobtusilactone A. To clarify whether caspases were the sole mediators for eliciting the observed apoptotic process, the effects of a broad caspases inhibitor, Z-VAD.fmk, was studied. Interestingly, Z-VAD.fmk was found to completely inhibit the isoobtusilactone A-induced oligonucleosomal DNA fragmentation, yet it could only prevent limited amount of cells from becoming apoptosis-prone. These data implied that other mechanism(s) might also be important factors and led us to study the possible involvement of apoptosis-inducing factor (AIF), a mediator arbitrating caspase-independent apoptosis, in isoobtusilactone A-induced apoptotic process. Our data indicated that isoobtusilactone A could elicit the nuclear translocation of AIF observed along with the occurrence of large-scale DNA fragmentation. Reduction of AIF expression by AIF-siRNA transfection suppressed large-scale DNA fragmentation. Interestingly, inhibition of AIF expression by AIF-siRNA did not prevent isoobtusilactone A-induced oligonucleosomal DNA fragmentation. When the cells were simultaneously treated with AIF-siRNA and Z-VAD.fmk, both large-scale DNA and oligonucleosomal DNA fragmentations were almost completely prevented. In conclusion, our data suggest that isoobtusilactone A induced apoptotic cell death was caused by the increase of ROS, followed by the disruption of mitochondrial transmembrane potential ( m), further mediated by both caspase-dependent and caspase-independent pathways. |
目次 Table of Contents |
中文摘要 ----------------------------------------------------------------------------- 1 英文摘要 ----------------------------------------------------------------------------- 3 第一章 緒論 ------------------------------------------------------------- 5 第二章 文獻探討 ------------------------------------------------------------- 6 第三章 材料與方法 -------------------------------------------------------------- 16 第四章 實驗結果 -------------------------------------------------------------- 31 第五章 討論與結論 -------------------------------------------------------------- 38 參考文獻 -------------------------------------------------------------------------------- 41 圖----------------------------------------------------------------------------- 49 表----------------------------------------------------------------------------- 76 附錄一 -------------------------------------------------------------------------------- 77 附錄二 -------------------------------------------------------------------------------- 78 |
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