臺灣博碩士論文加值系統

胰臟癌是西方國家是造成癌症死亡的第四項惡性腫瘤，在臺灣也是十大癌症死因之一。百分之八十五的胰臟癌在診斷確立時，已有遠處轉移或局部侵襲至臨近器官，此時只有少於百分之十能夠接受根除性手術治療。胰臟癌患者的平均存活時間只有四至六個月，並且整體的五年存活率小於百分之五。咖啡酸苯乙酯是蜂膠中所具有生物活性的成份。之前對咖啡酸苯乙酯的研究顯示咖啡酸苯乙酯是具有抗病毒、抗發炎、抗氧化、放射線治療的致敏劑等作用的性質。本實驗將以上列藥物作用於人類胰臟癌細胞株，希望能得到一些調控細胞週期及誘導細胞凋亡的相關機轉。對於中度及低度分化人類胰臟癌細胞株BxPC-3與PANC-1,我們以trypan blue dye exclusion test來偵測咖啡酸苯乙酯對細胞株生長的抑制作用。我們藉著觀察morphology，DNA histogram，annexin-V/PI雙染法，agarose gel electrophoresis來檢驗咖啡酸苯乙酯造成人類胰臟癌細胞株BxPC-3的死亡形式。隨著細胞株加入10 μg/mL濃度的咖啡酸苯乙酯處理，BxPC-3細胞株在第三天達到80.4 ± 4.1%的生長抑制，PANC-1細胞株在第三天達到74.3 ± 2.9%的生長抑制。BxPC-3細胞株在咖啡酸苯乙酯處理的第二天見到典型的細胞凋亡型態變化但並未見到明顯的DNA片斷化。隨著細胞株加入不同濃度5 μg/mL至20 μg/mL的咖啡酸苯乙酯處理而隨著時間增加sub-G1 population的趨勢，第2日起有較高的細胞株的sub-G1 population 增加。BxPC-3以10 μg/mL濃度的咖啡酸苯乙酯處理、處理的細胞株產生粒線體膜電位下降，在二天後呈現下降至未處理細胞的66.2 ± 11.4%。BxPC-3在經CAPE培養之前加入泛caspase抑制劑Z-VAD-fmk 50 μM處理，呈現部分的生長的抑制作用及sub-G1細胞群減少。並且BxPC-3細胞加入CAPE 10 μg/mL處理，在2小時後發現caspase-3/7的活性有2倍增加。說明caspase在咖啡酸苯乙酯誘導人類胰臟癌細胞株細胞凋亡中扮演相關的角色。本實驗結果顯示咖啡酸苯乙酯有強力的誘導人類胰臟癌細胞株細胞凋亡的活性，細胞死亡的同時也伴隨著粒腺體膜電位的減少及caspase的活化。

Pancreatic cancer is the fourth leading cause of cancer death in the western countries and the tenth in Taiwan. There are more than 85% of pancreatic cancers metastasized or extended locally at the time of diagnosis. Finally there were about less than 10% of the pancreatic cancer patients able to undergo curative resection. The average survival duration for the patients with pancreatic cancer is about 4–6 months, and the overall 5-year survival rate is less than 5%. An effective treatment or novel agent for this devastating disease is urgently needed. Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of honeybee propolis. The previous studies showed several biological functions, including anti-viral, anti-inflammatory, antioxidant and radiation sensitization effects of CAPE. This study was aimed to investigate the effect of caffeic acid phenethyl ester on inducing apoptosis in human pancreatic cancer cells. The growth inhibitory effect of the CAPE on BxPC-3 and PANC-1, moderate to poorly differentiated human pancreatic carcinoma cell lines, were estimated by trypan blue dye exclusion test. We examined the type of cell death in BxPC-3 after CAPE treatment by observation of morphology, DNA histogram, annexin-V/PI staining and DNA agarose gel electrophoresis. The CAPE treatment (10 μg/mL) resulted in marked growth inhibition on BxPC-3 cells up to 80.4 ± 4.1% and PANC-1 to 74.3 ± 2.9% at day 3. CAPE also induced characteristic morphological changes of apoptosis but there were no DNA fragmentation in BxPC-3 cell at day 2. Estimation of the apoptotic percentage showed a time-dependent increase after CAPE treatment. CAPE (10 μg/mL) induced a significant decrease in mitochondrial transmembrane potential to 66.2 ± 11.4% of the untreated level after 2 days. The growth inhibition and sub-G1 population was partially blocked by pretreatment with pan-caspase inhibitor Z-VAD-fmk (50 μM), which indicating a caspase-related mechanism involved in CAPE-induced apoptosis. The caspase-3/7 activity were approximately 2 times greater after treatment by caspases substrate activity assay. These results suggested that CAPE is a potent apoptosis-inducing agent and its action is accompanied by loss of mitochondrial transmembrane potential and activation of caspase.

目錄 I
圖表目錄 III
中文摘要 IV
英文摘要 V
緒論 1
研究方法 5
常用化學藥品
實驗儀器
細胞株
培養條件
細胞培養液
解凍細胞
冷凍細胞
化學物及處置
細胞存活率測定
偵測細胞凋亡的方法
形態學
細胞週期亞二倍體(sub-G1 population)之分析
以annexin-V-FITC/propidium iodide雙染法定量細胞凋亡
DNA 片斷化
細胞凋亡機轉的探討
粒腺體膜電位的改變
蛋白質定性與定量的分析
蛋白質的萃取
聚丙烯醯胺膠體電泳法
西方墨點法
計量有關調控凋亡的蛋白質(Bcl-2, Bax)
計量粒線體內有關調控凋亡的蛋白質
(Smac/Diablo, HtrA2/Omi, AIF, cytochrome c)
凋亡蛋白酵素caspase的活性測定
統計與分析
結果 21
細胞株的形態
BxPC-3與PANC-1細胞經不同濃度及時間CAPE處理的生長曲線
BxPC-3經不同濃度及時間CAPE處理的生長抑制率
PANC-1經不同濃度及時間CAPE處理的生長抑制率
處置前後形態上的改變
BxPC-3經不同濃度及時間CAPE處理的sub-G1細胞週期之分析
以Annexin-V-FITC/propidium iodide雙染法偵測BxPC-3經不同濃度及時間CAPE處理的細胞凋亡率
DNA 片斷化
BxPC-3經不同濃度及時間CAPE處理的粒腺體膜電位的改變
使用西方墨點法計量細胞漿(Bcl-2，Bax)及粒線體內(Smac/Diablo，HtrA2/Omi，AIF，cytochrome C)有關調控凋亡的蛋白質
BxPC-3經不同濃度CAPE及泛caspase 抑制劑處理的生長抑制率
BxPC-3經不同濃度CAPE及泛caspase抑制劑處理的sub-G1細胞週期之分析
BxPC-3經CAPE處理後的caspase family 的活性測定
討論 26
結論與展望 35
參考文獻 34
圖表 41

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