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研究生:高士傑
研究生(外文):Shih Chieh Kao
論文名稱:以動態磷酸化蛋白體學探討丹參酮IIA作用於胃癌AGS細胞的反應路徑
論文名稱(外文):Temporal Phosphoproteome Dynamics Reveal Response Pathways of Tanshinone IIA in Gastric Cancer AGS Cells
指導教授:阮雪芬阮雪芬引用關係
指導教授(外文):Hsueh-Fen Juan
口試委員:黃宣誠李岳倫陳頌方王憶卿
口試委員(外文):Hsuan-Cheng HuangYueh-Luen LeeSung-Fang ChenYi-Chin Wang
口試日期:2015-07-28
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子與細胞生物學研究所
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:2015
畢業學年度:103
語文別:英文
論文頁數:123
中文關鍵詞:丹參酮胃癌動態磷酸化蛋白體技術磷酸化蛋白體蛋白質折疊
外文關鍵詞:Gastric cancerTanshinone IIA (TIIA)Time-Dependent PhosphoproteomicsProtein unfolding response
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胃癌是常見的致死癌症之一,然而到現在仍然沒有一個有效的治療方法或是藥物可以對付胃癌,所以尋找一個安全且可以治療胃癌的方法或藥物是我們的首要目標。在先前的研究中,丹參酮IIA來自於天然的中草藥<丹參>萃取物,具有抗癌的功能,在許多不同的癌症中它都具有相當好的功效,因此丹參酮IIA是相當具有潛力的藥物。然而目前的研究對於丹參酮IIA抗癌的整體反應途徑仍尚未清楚。在本篇研究中我們利用動態磷酸化蛋白體技術揭露丹參酮IIA在胃癌AGS細胞中的調控及訊息傳遞路徑。在本次實驗中我們偵測到3,332個磷酸胜肽,辨認出1,092個磷酸蛋白質,具有3,615個鄰酸化位置,表現量有差異的磷酸位置和磷酸蛋白分別為349個和220個,我們進而利用生物資訊學分析這些表現量有顯著差異的磷酸化蛋白質。結果顯示丹參酮IIA在短期會影響胃癌細胞的訊息核糖核酸的處理、引發去氧核醣核酸的受損和蛋白質未折疊或折疊錯誤。此外,去氧核醣核酸的受損及蛋白質未折疊或折疊錯誤發生在丹參酮IIA長時間處理的細胞中。這些結果顯示丹參酮IIA的反應傳遞途徑可能是跟這兩者有關。最後,我們利用西方墨點法證實丹參酮IIA影響蛋白質折疊。本篇研究利用動態磷酸化蛋白體技術去探討丹參酮IIA在胃癌細胞的抑制效果,希望本篇研究可以對胃癌的治療方向或是策略帶來新的曙光。

Gastric cancer is one of the most common causes of cancer deaths worldwide. Since the treatment strategies for gastric cancer are controversial, development of efficient therapeutic agents is important for gastric cancer. Tanshinone IIA (TIIA) is a diterpene quinone extracted from the traditional herbal medicine, Danshen (Salvia miltiorrhiza), and has been reported to have anti-tumor potential against several cancers. We previously found that TIIA could kill gastric cancer AGS cells. However, the signaling changes underlying TIIA-treated gastric cancer cells are still unclear.
In this study, we applied a time-dependent phosphoproteomic approach to reveal the regulatory effects of TIIA in AGS cells. Our results showed that a total of 1092 phosphoprotiens and 3332 phosphopeptides were identified in 3615 phosphorylation sites and 349 phosphosites corresponding to 220 phosphorylated proteins were significantly regulated. By using bioinformatics analysis, we found that TIIA might regulate several cellular processes of gastric cancer cells, such as transcription mRNA processing, DNA damage and protein unfolding responses. We also reveal time course of the biological processes with TIIA treatment in AGS cells. In addition, DNA damages and protein unfolding response still occur in AGS cells for the long-termed period. Finally, we further validated that TIIA may cause the protein unfolding response and DNA damage via inducing ROS production. These findings not only uncover the molecular mechanisms mediated by TIIA but also contribute to the development of gastric cancer therapy.


誌謝 I
中文摘要 II
ABSTRACT III
Contents V
List of Figures VII
List of Tables VIII
Chapter 1 Introduction 1
1.1 Gastric cancer 1
1.2 Tanshinone IIA 1
1.3 Phosphoproteiomics 3
Chapter 2 Materials and methods 5
2.1 Cell culture 5
2.2 Drug preparation and treatment of cultured cells 5
2.3 Sample preparation for cell phosphoproteome 5
2.4 Dimethyl labeling of peptides 6
2.5 Phosphopeptide enrichment 7
2.6 SCX fractionation 8
2.7 NanoLC–MS/MS analysis 9
2.8 Quantitation and identification for phosphoproteomic data 9
2.9 Bioinformatics analysis 10
2.10 Western blot 12
2.11 Detection of reactive oxygen species by Flow cytometry 13
Chapter 3 Results 14
3.1 Quantitative analysis of dynamic phosphorylation profiles of gastric cancer cells treated wiht Tanshinone IIA (TIIA) 14
3.2 Functional enrichment and network analysis of TIIA induced differentially regulated phosphoproteins 15
3.3 TIIA induces DNA damage and protein unfolding response to apoptotic process and cell death in temporal dynamics. 17
3.4 TIIA induces protein unfolding response in temporal dynamics and long term period. 17
3.5 TIIA induces the protein unfolding response through ROS 19
Chapter 4 Discussion 20
Chapter 5 Conclusions 23
Chapter 6 Future work 24
References 25
Figures 31
Tables 48

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