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研究生:邱家蓁
研究生(外文):Chiu, Jia-Jen
論文名稱:探討牛磺酸與維生素C作為肺癌細胞株暴露Benzo [α] Pyrene (BaP)之預防效果
論文名稱(外文):Benzo [α] Pyrene (BaP)之預防效果Taurine and Vitamin C Play as Antioxidants to Prevent Benzo [α] Pyrene (BaP) Attack in Lung Cancer Cell
指導教授:黃登福黃登福引用關係
指導教授(外文):Huang, Deng-Fwu
口試委員:陳泰源周薰修鄭森雄
口試委員(外文):Chen, Tai-YuanChou, Shin-shouJeng, Sen-Shyong
口試日期:2016-06-14
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:98
中文關鍵詞:多環芳香烴化合物苯并芘牛磺酸維生素C
外文關鍵詞:Polycyclic aromatic hydrocarbonsBenzo [α] PyreneTaurineVitamin C
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肺癌 (Lung cancer) 的發病率占全世界癌症人口的 29%,而燒烤食物以及香菸中的主成分多環芳香烴PAHs (Polycyclic aromatic hydrocarbons) 如苯比芘 (Benzo [α] pyrene; BaP) 被認為與肺部癌病變有著重要的關聯性。PAHs 為石油、焦炭等不完全燃燒後的產物,其暴露途徑以菸草煙霧、空氣汙染、加工食品、以及皮膚接觸等較為常見。本報告以人類肺泡上皮癌細胞株 A549 (Human lung adenocarcinoma epithelial cell line) 探討暴露雜環胺類化合物 BaP 之毒性作用。BaP 以 2-50 μM 暴露 A549 細胞 24-72 hr 後檢測其細胞毒性,進一步探討 BaP 誘導細胞凋亡之路徑。即探討調控細胞凋亡路徑中半胱胺酸蛋白酶 (Cysteine dependent aspartate-specific protease) 與凋亡相關蛋白如抗凋亡蛋白 B-cell lymphoma 2 (Bcl-2)、B-cell lymphoma-extra large (Bcl-xl) 與促凋亡蛋白 Bcl-2-antagonist / killer 1 (Bak)、Bcl-2-associated x protein (Bax) 以及凋亡路徑下游 cytochrome c (cyt c) 與 Phospho-NF-kB p65 (Ser276) 之蛋白質表現量,以流式細胞儀檢測細胞內活性氧分子 (Reactive oxygen species; ROS)、粒線體膜電位 (Mitochondrial membrane potential; MMP; ΔΨ) 生成量,輔助分析細胞因暴露 BaP 造成氧化損傷及細胞毒性具相關性。
第一部分結果顯示受 BaP 暴露後,細胞藉由氧化還原循環而誘導氧化自由基 (Free radical-oxygen species; free-ROS) 轉換為具活性之自由基,透過 MMP 的改變,造成內質網壓力 (Endoplasmic reticulum stress; ER stress) 增加而導致細胞走向凋亡 (Apoptosis)。其細胞凋亡機制為 A549 細胞隨 BaP 暴露濃度的上升,使凋亡蛋白 Bax、Bak 增加,抗凋亡蛋白 Bcl-2、Bcl-2 降低,凋亡路徑相關之 Cytochrome c 及 caspase 3/7 活性與 NF-κB 蛋白隨暴露劑量而增加,進而導致細胞週期中 GO/G1 phase 延滯並使細胞核內的 DNA 產生片斷化情形。
第二部分將細胞暴露 BaP 同時添加 Taurine 與 Vitamin C 25 mM,作為探討暴露 BaP (2-50 μM) 於 A549 細胞之抗氧化以及化學預防之效用。結果顯示,透過抗氧化劑的添加可使 ΔΨ 增加,ROS 表現量下降以及下游 caspase 3/7 的活性降低,改善因暴露 BaP 造成細胞週期中的 GO/G1 phase 延滯情形,使 GO/G1 phase 縮短,進一步減少促凋亡蛋白 Bax 和 Bak 的表現,並使抗凋亡蛋白 Bcl-2 和 Bcl-xl 表現量增加,造成細胞凋亡路徑相關之 Cytochrome c 之表現量降低,又 NF-κB 活性增加,顯示添加抗氧化劑可減緩細胞因暴露 BaP 所造成之損傷,並且其抑制細胞凋亡機制與 NF-κB 有關。

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[α]pyrene (B[α]P; BaP), is a potent lung carcinogen derived from tobacco smoking and food. BaP also has latent capability to activate caspase activity for inducing cell death. Taurine and vitamin C have potent antioxidative activities that protect cells from oxidative stress and cellular damage. The objectives of the present study were to investigate the adverse effects of BaP on human pulmonary epithelial cell line (A549), the potential protective effects of taurine and vitamin C against BaP-induced cellular damage, and the molecular mechanisms of actions. Therefore, A549 cells were exposed to different doses of Bap (2-50 μM) for determining direct toxic effects on the cell apoptosis . It is found that BaP induces apoptosis in a p53-mediated and caspase 3 and caspase 7 dependent manner, which relate to the accumulation of the G0/G1 phase of the cell cycle. BaP treatment resulted in a significant decrease in the protein expression of Bcl-2 and Bcl-xl, whereas protein expression of Bax and Bak and cytochrome c activity were significantly increased. Further, A549 cells were induced a significant increase in the activities of caspase 3 and caspase 7 as well as the number of apoptotic cells by BaP exposure. BaP also caused oxidative stress by increasing reactive oxygen species (ROS) production and reducing mitochondrial membrane potential (MMP). Meanwhile, A549 cells were damaged and induced to produce genomic DNA ladder after exposing BaP. Taurine and vitamin C prevented cells from BaP- induced cell cycle arrest and growth inhibition, and significantly suppressed DNA fragmentation. Taurine and vitamin C significantly restored survival of BaP exposed cells. Taurine and vitamin C could reverse some of these BaP-mediated alterations and therefore be effective natural compounds against the adverse effects of BaP in lung cells.
目錄
摘要...............................................................................................................................II
英文摘要......................................................................................................................III
壹、 文獻整理................................................................................................................1
一、 肺癌.............................................................................................................. 2
二、 多環芳香烴化合物........................................................................................3
三、 抗氧化物質....................................................................................................9
四、細胞週期的調控 ….11
貳、研究內容 23
第一章、 BaP暴露於人類肺癌細胞A549造成細胞凋亡之途徑探討..........21
一、 前言 21
二、 材料與方法 22
三、 結果 32
四、 討論……………………………………………………………………….36
五、 圖表……………………………………………………………………….38
第二章、抗氧化物維生素C與牛磺酸對於BaP造成細胞損傷之預防效果探討...47
一、 前言 47
二、 材料與方法 48
三、 結果 61
四、 討論……..………………………………………………..………….……65
五、 圖表…………………………………………………………………..……66
結論..…………………………………………………………………………………82
參考文獻 ……………………………………………………………………………..84
謝辭…………………………………………………………………………………97
附錄一..........................................................................................................................98


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