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系統識別號 U0026-0102201300225300
論文名稱(中文) 奎寧抑制RAGE-STAT3調控之自體吞噬路徑以促進紫檀芪誘發之胰臟癌細胞死亡
論文名稱(英文) Chloroquine Promotes Pancreatic Cancer Cell Apoptosis Induced by Pterostilbene Through Downregulation of RAGE-STAT3 Mediated Autophagy Pathway
校院名稱 成功大學
系所名稱(中) 環境醫學研究所
系所名稱(英) Institute of Environmental and Occupational Health
學年度 101
學期 1
出版年 102
研究生(中文) 呂懿臻
研究生(英文) Yi-Jhen Lyu
學號 s76991102
學位類別 碩士
語文別 中文
論文頁數 61頁
口試委員 口試委員-郭靜娟
口試委員-潘敏雄
口試委員-何元順
指導教授-王應然
中文關鍵字 紫檀芪  奎寧  胰臟癌  自體吞噬 
英文關鍵字 Pterostilbene  Chloroquine  pancreatic cancer  autophagy 
學科別分類
中文摘要 胰臟癌在美國癌症相關死因排名第四,五年存活率低於5%。胰臟癌常用的治療方法是以Gemcitabine (GEM)做為標準藥物,可是胰臟癌對GEM有高度抗性。先前的研究表明胰臟癌暴露於GEM會透過NF-κB 增加RAGE的表現,而過度表現的RAGE會誘導自體吞噬的現象而增加腫瘤細胞的存活和化學治療的抗性。紫檀芪屬於芪類 (stilbenoid),與白藜蘆醇相似,實驗證明紫檀芪有預防或治療癌症的潛力,並且會誘導腫瘤細胞的細胞凋亡或自體吞噬反應。奎寧為一種安全且有效的抗瘧疾藥物,也被證實具有抑制自體吞噬的能力。本實驗目的為使用臨床用藥奎寧來抑制胰臟癌細胞的自體吞噬以促進紫檀芪誘導的細胞死亡,進而達到治療胰臟癌的效果,且探討兩種藥物合併所造成BxPC-3和Mia PaCa-2兩株胰臟癌細胞死亡的分子機轉。在細胞實驗部分,利用人類胰臟癌細胞株BxPC-3和MiaPaCa-2觀察紫檀芪和奎寧兩藥物合併處理所誘導的分子機制與訊息調控路徑。以MTT分析細胞存活率,使用流式細胞儀分析細胞週期、細胞凋亡、自體吞噬,利用acridine orange染色觀察細胞自體吞噬酸性小泡的產生,並以西方墨點法分析相關調控蛋白。在動物實驗方面,藉由原位癌模式(orthotopic model)搭配活體分子影像系統來評估紫檀芪合併奎寧是否具有治療的效果,並進一步使用西方墨點法及組織免疫染色確認合併治療胰臟癌的機轉。在細胞實驗中發現,單獨處理紫檀芪時,MiaPaCa-2相較於BxPC-3的死亡率低,且觀察到MiaPaCa-2有較高量的細胞自噬作用。利用奎寧抑制紫檀芪所造成MiaPaCa-2的自體吞噬後,發現死亡率有顯著性的提高,而使用流式細胞儀也測得細胞凋亡有上升的現象。在蛋白質表現量方面,藥物合併處理胰臟癌細胞後,兩株細胞皆可觀察到RAGE、p-STAT3和p-AKT蛋白受到抑制。而動物實驗證實口服紫檀芪合併奎寧使用,可減少胰臟原位癌腫瘤重量及體積,達到抑制腫瘤生長的效果。結果說明胰臟癌細胞單獨處理紫檀芪可經由誘導細胞凋亡來達到抑制生長的作用,並藉由奎寧抑制細胞內的RAGE-STAT3路徑進而抑制自體吞噬來加強紫檀芪所誘導的細胞凋亡。
英文摘要 Pancreatic cancer is reported to be the fourth leading cause of cancer death in the United States with a 5-year survival less than 5%. The common treatment is the Gemcitabine (GEM) therapy for pancreatic cancer, but pancreatic tumors are highly resistant to GEM. Previous studies have demonstrated that exposure of pancreatic tumor cells to GEM provoked a nuclear factor kappa B (NF-κB) -dependent increase in RAGE expression, and overexpression of RAGE led to greater induction of autophagy that improved tumor cell survival and chemotherapeutic resistant. Pterostilbene is a stilbenoid chemically related to resveratrol. Experimental evidence shows that Pterostilbene has potential for the prevention or treatment of cancers by inducing apoptosis or autophagy in cancer cells. Chloroquine is an effective and safe anti-malarial agent, and has the ability to inhibit autophagy. The aim of this study is to investigate whether pterostilbene combined with autophagy inhibitor Chloroquine could potentiate cell death in pancreatic cancer cells and to study the molecular anti-cancer mechanisms of pterostilbene combined with chloroquine. Cell viability was determined by MTT assay. Cell cycle, apoptosis, and autophagy were analyzed by flow cytometry. The expression of proteins that regulate apoptosis and autophagy were performed by western blotting. In in vivo study, we use orthotopic model with IVIS image system to evaluate the anti-cancer effects of pterostilbene combined with chloroquine, and further to demonstrate the related mechanisms by western blotting and immunohistochemistry. In the in vitro studies, the cell viability in MiaPaCa-2 cells were higher than BxPC-3 cells after treatment with pterostilbene. The results indicated that MiaPaCa-2 had higher autophagy. Autophagy inhibitions by chloroquine in MiaPaCa-2 significantly decreased the cell viability, and increased apoptosis that induced by pterostilbene through down regulation of RAGE、STAT3 and AKT pathways. In the in vivo experiment, pterostilbene combined with Chloroquine has demonstrated to inhibit orthotopic pancreatic cancer growth by reducing tumor weight and tumor volume. The results indicated pterostilbene can inhibit pancreatic cancer cells growth by inducing apoptosis. Autophagy suppression by chloroquine potentiates pterostilbene induced cell death in pancreatic cancer cells through inhibiting RAGE-STAT3 pathway and AKT pathways.
論文目次 第一章、 序論 3
第二章、 文獻回顧 4
第一節、胰臟癌 (Pancreatic cancer) 及其治療方式與細胞死亡 4
第二節、RAGE和STAT3調控細胞自噬促進胰臟癌細胞的存活 7
第三節、天然物紫檀芪在癌症化學預防與癌症治療的角色 9
第四節、奎寧 (Chloroquine) 12
第三章、 研究目的 14
第四章、 研究架構 15
第一節、 In vitro: 15
第二節、 In vivo: 16
第五章、 研究材料與方法 18
第一節、研究材料 18
第二節、研究方法 25
In vitro 25
(一) 細胞培養 25
(二) 細胞解凍 25
(三) 細胞繼代培養 25
(四) 細胞冷凍 26
(五) 細胞計數 26
(六) MTT assay 26
(七) 細胞週期分析 27
(八) 細胞凋亡與壞死分析 27
(九) 自體吞噬分析 27
(十) 螢光顯微鏡分析自體吞噬現象 28
(十一) 穿透式電子顯微鏡(TEM) 28
(十二) 西方墨點法 29
In vivo 30
統計分析 33
第六章、 實驗結果 34
第一節、紫檀芪對於人類胰臟癌細胞之細胞毒性的劑量效應 34
第二節、分析紫檀芪對BxPC-3和MiaPaCa-2細胞死亡現象 35
第三節、探討奎寧合併紫檀芪處理BxPC-3與MiaPaCa-2細胞的效果 36
第四節、以電子顯微鏡觀察其細胞死亡方式 37
第五節、人類正常胰臟細胞與四株人類胰臟癌細胞蛋白表現的差異 38
第六節、紫檀芪與奎寧對於人類胰臟癌細胞之訊息路徑相關蛋白的表現 38
第七節、胰臟原位癌異體移植活體動物模式 39
第八節、觀察在動物實驗中合併紫檀芪與奎寧所影響的細胞凋亡現象 40
第七章、 討論 41
第八章、 結論與建議 45
第九章、 參考文獻 46
圖表 51
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