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研究生:張競文
研究生(外文):Ching-Wen Chang
論文名稱:磷脂氫麩胱甘肽過氧化酵素PHGPx抑制砷化鈉和過氧化氫所誘導人類上皮癌細胞A431凋亡之機制
論文名稱(外文):Inhibition of sodium arsenite and hydrogen peroxideinduced apoptosis by phospholipid hydroperoxide glutathione peroxidase (PHGPx) in human epidermoid carcinoma A431 cells
指導教授:陳清浚
指導教授(外文):Ching-Jiunn Chen
學位類別:碩士
校院名稱:中華醫事學院
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:94
語文別:中文
論文頁數:99
中文關鍵詞:磷脂氫麩胱甘肽過氧化酵素過氧化氫砷化鈉A431
外文關鍵詞:Hydrogen peroxidesodium arsenitePHGPx
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許多活性氧物質都屬含氧自由基,這些自由基會造成一些氧化性傷害,舉凡老化、癌症都被研究報導出與自由基攻擊細胞與基因有關。細胞內屬於抗氧化酵素之一的PHGPx能特異性地代謝磷脂氫過氧化物,而致環氧酵素,十二脂氧酵素和十五脂氧酵素的酵素活性降低。
砷化鈉(sodium arsenite; NaAsO2)是一種環境致癌毒物,目前對其致癌的機轉仍不清楚。 之前的研究指出As3+的致癌性可能與氧化壓力的產生有關。
過氧化氫(Hydrogen peroxide; H2O2)為一種強氧化性的物質,在生物體中可經由生長因子刺激或粒腺體的呼吸鏈所產生,研究發現大量H2O2可促進細胞凋亡但少量時卻可抑制細胞凋亡。
本篇的研究主要在探討人類子宮頸上皮癌細胞(A431 cells)的PHGPx含量是否可對抗高劑量As3+或H2O2刺激下所引發的細胞傷害。我們先透過基因轉殖的方式建立出PHGPx含量不同之穩定型細胞株,再由caspase-3活化以及DNA fragmentation 的結果發現,大量表現PHGPx之細胞株在As3+或H2O2刺激下比控制組更能抵抗細胞凋亡;反之,經由siRNA抑制PHGPx蛋白表現的細胞株,則較控制組容易產生細胞凋亡。此外,我們也發現在As3+刺激之下,細胞走向凋亡是透過活化caspas-8(外在路徑) 與 caspase-9(內在路徑);但H2O2刺激之下,細胞僅活化caspas-8(外在路徑)而已。因此,我們的結論是: PHGPx可藉由抑制內外路徑的活化而保護細胞免於受到多種刺激物所誘導之細胞凋亡。
Many of reactive oxygen species that belong to oxygen radicals can lead cells in an oxidative damage. It has been reported that some phenomenons such as aging or tumorigenesis are associated with the damage of oxygen radicals. PHGPx belongs to a kind of antioxidant enzyme in cells that may inhibit the reaction of oxygenation by removing hydroperoxides and decrease the enzyme activity of arachidonate metabolism.
Sodium arsenite (NaAsO2), a potent human carcinogen, has not been clearly understood what its carcinogenesis is. It has been suggested that the carcinogenesis of arsenite is associated with oxidative stress.
Hydrogen peroxide (H2O2), a potent oxide, can be produced by way of growth factors or respiratory chain of mitochondria in organism. It has been reported the high dose of H2O2 can induce cell apoptosis, oppositely, low dose of H2O2 can inhibit apoptosis.
In this study, we focused on whether PHGPx could protect cells
from the damage of high dose H2O2 or As3+ in human epidermal
carcinoma (A431) cells. First, we established a serial of stable
transfectants with differential level of PHGPx expressing. We
followed up to observe the appearance of apoptosis with caspase-3
activation or DNA fragmentation under various stimulators. A
transfectant with overexpressing tag-PHGPx was more resistant to
apoptosis than vector control line under highly concentration of
As3+ or H2O2 treatment. In contrast, a transfectant which PHGPx was
knockdown by siRNA was more sensitive to apoptosis than vector
control line under similar treatment. In addition, we also found that the activation of apoptosis inducing by As3+ was mediated through both caspase-8 (Extrinsic pathway) and caspase-9 (Intrinsic
pathway) signaling pathways, but only caspase-8 signal pathway by
H2O2. Consequently, we concluded that PHGPx could inhibit the
activation not only the extrinsic but also the intrinsic signal
pathway in cells, and follow to protect cell from apoptosis under
various stimulators.
口試委員會審定書………………………………………………………………………………..
授權書…………………………………………………………………………………………..ii
誌謝……………………………………………………………………………………………..iii
中文摘要………………………………………………………………………………………..iv
英文摘要………………………………………………………………………………………..vi
目錄…………………………………………………………………………………………….viii
圖目錄…………………………………………………………………………………………...x
第一章 緒論
砷的種類與文獻回顧…………………………………………………………………………….1
砷的代謝與細胞毒性…………………………………………………………………………….2
過氧化氫的簡介與文獻回顧…………………………………………………………………….4
過氧化氫的代謝………………………………………………………………………………….5
磷脂氫麩胱甘肽過氧化酵素(PHGPx)的作用………………………………………………….7
研究動機與實驗目的…………………………………………………………………………….15
第二章 材料與方法………………………………………………………………………….19
第三章 實驗結果
PHGPx大量/降低表現之細胞株製備…………………………………………………………..32
細胞株表現PHGPx之生長結果比較…………………………………………………………….34
PHGPx大量表現可抑制砷化鈉所誘導人類上皮癌細胞凋亡……………………………….35
PHGPx大量表現抑制過氧化氫所誘導人類上皮癌細胞凋亡………………………………….38
第四章 討論與結論………………………………………………………………………….41
參考文獻 ……………………………………………………………………………………….50
圖表……………………………………………………………………………………………….65
附錄……………………………………………………………………………………………...91
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