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研究生:黃永豊
研究生(外文):Yung-Feng Huang
論文名稱:香豆精(Coumarin)抑制人類子宮頸癌細胞株(HeLa)增生並誘發細胞凋亡之機轉
論文名稱(外文):Coumarin inhibits proliferation of human cervical cancer cells via inducing cell cycle arrest and apoptosis
指導教授:莊淨媛鍾景光鍾景光引用關係
指導教授(外文):Jing-Yuan ChuangJing-Gung Chung
學位類別:碩士
校院名稱:中國醫藥大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:72
中文關鍵詞:香豆精人類子宮頸癌細胞株細胞週期停止細胞凋亡
外文關鍵詞:coumarinHuman cervical cancer cellscell cycle arrestapoptosis
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本篇研究為利用天然植物中的香豆精(coumarin)處理人類子宮頸癌細胞株(HeLa)的作用機轉。主要探討目的如下:(1)香豆精是否能抑制子宮頸癌細胞(HeLa)增生。(2)以香豆精處理子宮頸癌細胞是否會誘發細胞凋亡(apoptosis)的現象。(3)以香豆精處理子宮頸癌細胞是否會造成細胞週期停止(cell cycle arrest)的現象。(4)以香豆精處理子宮頸癌細胞對於活性氧化物(reactive oxygen species)、粒線體膜電位改變(mitochondrial membrane potential)、鈣離子(Ca2+)產生的影響。(5)以香豆精處理子宮頸癌細胞造成細胞凋亡和細胞週期停止的機制。以MTT assay和流式細胞計數儀來偵測細胞的存活率,發現隨著藥物濃度的上升和加藥時間的延長,確實會抑制細胞存活率。以倒立式位相差顯微鏡來觀察細胞型態上的改變,結果發現有細胞型態不完整、細胞膜皺縮、與空泡化的現象。以流式細胞計數儀來偵測細胞週期,發現在G0/G1期的細胞比例上升,故香豆精會造成細胞週期的G0/G1期的停止,並有細胞凋亡(apoptosis)的現象產生。以流式細胞計數儀來偵測活性氧化物(reactive oxygen species),發現ROS有增加的趨勢,故推測香豆精會誘發細胞產生ROS。以流式細胞計數儀來偵測粒線體膜電位(mitochondrial membrane potential),發現粒線體膜電位下降,故推測香豆精會誘發細胞膜孔道的打開,而導致粒線體膜電位下降。以流式細胞計數儀來偵測鈣離子(Ca2+)濃度,發現鈣離子(Ca2+)濃度升高,故推測香豆精會經由粒線體釋出Ca2+進入內膜,造成鈣離子(Ca2+)濃度升高。以慧星試驗偵測細胞的DNA損傷(damage),發現在高濃度香豆精處理後,造成細胞拖尾的比例最為明顯,表示有DNA損傷的現象。而以DNA電泳膠片觀察香豆精對於人類子宮頸癌細胞株HeLa細胞的DNA情形,發現DNA斷裂呈階梯狀片段,表示有細胞凋亡的現象。而後以西方墨點法(Western blotting)來偵測蛋白表現量的改變,發現caspase-3、Cdk 4、p53、p21、Bax、Cytochrome c與iNOS蛋白表現量增加,而Cyclin D、CDK 2、CDC 25A、p15、Bcl-2、Bcl-xl、NF-κB p50、NF-κB p65蛋白表現量減少。綜合以上結果發現:以香豆精處理人類子宮頸癌細胞株(HeLa),在細胞內會產生ROS,導致DNA損傷,進而活化p53,之後活化p21,而p21會造成CDC 25A下降,進而抑制Cyclin D、Cdk2的表現,而p15亦會抑制Cyclin D,使細胞停止在G0/G1期。當p21活化後會使得粒線體內的Bcl-2家族蛋白減少,包括Bcl-2與Bcl-xl,並刺激粒線體Bax增加,此時粒線體的能量發生變化,粒線體會釋出鈣離子(Ca2+),並造成膜電位下降,而粒線體亦會釋出cytochrome c,並活化下游的caspase-3,造成DNA fragment,並誘發細胞凋亡,最終造成細胞死亡,進而抑制細胞的存活率。
In the last years, the strategy of selectively killing tumor cell by the induction of apoptosis has been addressed extensively. In this study, we have addressed the cytostatic and apoptosis effects of coumarin on human cervical carcinoma cell lines (HeLa). Treatment of HeLa cells with coumarin resulted in the inhibition of cell proliferation , arrest of cell cycle in G0/G1 phase and commitment to apoptosis. Inhibition of cell proliferation was examined by MTT assay and flow cytometry. The inhibition effect is dose-dependent manner. The effects on cell cycle phases were determined at 100 μM of coumarin using flow cytometry. The same concentration were used to study apoptosis, detected by morphological cell changes, sub-G1 peak detection and ladder fragmentation occurrence. Coumarin also induced the levels of Ca2+ concentration and decreased the levels of mitochondria membrane potential in HeLa cells. Moreover, the redox events associated with this compound and the protein levels of molecules involving cell cycle regulation and apoptosis have been investigated .The production of reactive oxygen species was slightly increased only after 30 min of treatment with 100 μM coumarin, reaching a maximum at 36 hr. The results also demonstrated that coumarin down-regulate the expression of cell cycle progress factors cyclin D, Cdc25A and CDK2.The coumarin up-regulate the expression of cell cycle progress factors cytodhrome c,p53 and p21.The active form of apoptosis key enzyme caspase-3 was increased and the apoptosis inhibitors Bcl-2 and Bcl-xl were down-regulated.The apoptosis inducer Bax were up--regulated.
Overall, the results suggest a pivotal role for oxidative stress in coumarin-induced apoptosis and, taking into account that tumor cells are deficient in antioxidants, suggest a plausible utilization of this compound as an anti-proliferative agent in cancer therapy.
目錄
頁次
中文摘要 1
英文摘要 2
致謝 3
目錄 4
圖表目錄 6
縮寫表 9
第一章 前言 10
第一節 研究緣起 10
第二節 研究目的 10
第二章 文獻探討 11
第一節 天然中草藥之應用 11
第二節 香豆精(coumarin)簡介 11
第三節 細胞週期的調控 12
第四節 細胞凋亡(Apoptosis) 15
第五節 活性氧化物(Reactive oxygen species) 17
第六節 粒腺體膜電位(Mitochondrial membrane potential)、鈣離子(Ca2+)與DNA
損傷(DNA damage) 19
第三章 研究設計與研究假說 21
第一節 研究設計 21
第二節 研究假說 21
第四章 研究材料與方法 22
第一節 實驗材料 22
第二節 實驗方法 24
第五章 實驗結果 29
第一節 香豆精在人類子宮頸癌細胞株對細胞增生的影響與細胞型
態上的改變情形 29
第二節 香豆精在人類子宮頸癌細胞株對細胞週期(Cell cycle)的影響 33
第三節 檢測香豆精在人類子宮頸癌細胞株細胞是否產生活性氧化物
(Reactive oxygen species) 35
第四節 香豆精在人類子宮頸癌細胞株細胞對膜電位(Mitochondrial
membrane potential)的影響 39
第五節 香豆精對細胞內鈣離子(Ca2+)濃度之影響 42
第六節 利用慧星試驗(Comet assay)觀察香豆精對於人類子宮頸癌
細胞株HeLa細胞其DNA傷害程度 46
第七節 利用DNA電泳膠片觀察香豆精對於人類子宮頸癌細胞株HeLa細胞其DNA ladder情形 47
第八節 利用西方墨點法(Western blotting)檢測香豆精對細胞週期蛋白表現量的影響 48
第九節 利用西方墨點法檢測香豆精在人類子宮頸癌細胞中對caspase蛋白與細
胞凋亡調控蛋白的表現量之影響 51
第十節 利用西方墨點法對香豆精在人類子宮頸癌細胞中Death Receptor與
iNOS蛋白表現量的變化 55
第六章 討論 58
第七章 結論與建議 63
第一節 結論 63
第二節 建議 63
參考文獻 65
自述 72
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