博碩士論文 992413001 詳細資訊



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姓名 曾愛倫(Ai-Lun Tseng)  查詢紙本館藏   畢業系所 系統生物與生物資訊研究所
論文名稱 中國傳統醫藥蒙古黃耆在HCT116結腸癌細胞體外和體內實驗呈現腫瘤抑制作用
(Traditional Chinese Medicine Astragalus membranaceus (Fischer) Bge. var. mongolicus (Bge.) Hsiao Exhibits Tumor Inhibitory Effect in HCT116 Colorectal Cancer Cells in vitro and in vivo)
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摘要(中) 許多腫瘤細胞在治療過程中會去抵抗化療藥物所產生的細胞毒性，因此，有需要重新審視傳統中草藥於大腸直腸癌的治療。此研究利用黃耆萃取物治療HCT116大腸癌細胞的研究發現黃耆確實具有抗腫瘤生長的功能。在動物實驗中，我們發現經過黃耆治療的小鼠腫瘤體積較對照組有顯著縮小。藉由基因晶片圖譜分析後，發現表觀基因體與代謝等相關的功能產生變化。另外，在微核糖核酸分析中，許多標靶微核糖核酸也參與代謝及細胞內運輸有關之功能。此研究也運用Library of Integrated Network-based Cellular Signatures資料庫找出與黃耆作用相似的天然藥物。經黃耆治療後的異種移植小鼠血清中使用酵母菌蛋白質晶片偵測其蛋白質表現量也不同於對照組，並發現免疫相關的生物標記物在血液中具有顯著差異。最後，本實驗室也建立一組對5-氟尿嘧啶抗藥的HCT116細胞株。這些細胞株不只呈現多倍體的型態，而在5-氟尿嘧啶與黃耆合併治療中也能抑制細胞存活率。我們的研究結果發現黃耆對於大腸直腸癌具有潛在的治療作用，希望此研究將提供其影響性在藥廠等商業應用上。
摘要(英) The fact that many chemotherapeutic drugs cause chemoresistance and side effects during the course of treating colorectal cancer necessitates development of novel cytotoxic agents aiming to attenuate new molecular targets. Here, we show that Astragalus membranaceus (Fischer) Bge. var. mongolicus (Bge.) Hsiao (AM), a traditional Chinese medicine, can inhibit tumor growth in vitro and in vivo thus the underlying molecular mechanisms are elucidated. The effects of AM on HCT116 cell viability and apoptotic events were studied over a dose range of 0-500 μg/ml. The viability assay showed that AM-treated cells were significantly lower than control. In the animal model, the mice were treated with either water or 500 mg/kg AM once per day, before being sacrificed for extraction of tumors and serum, which were then subjected to microarray expression profiling. The identified genes as differentially expressed between treated mice and controls revealed that administration of AM suppresses chromosome organization, histone modification, regulation of macromolecule metabolic process and others. A separate analysis focused on differentially expressed microRNAs revealed involvement of macromolecule metabolism, intracellular transport, etc. and implicated several cancer signaling pathways. For validation, inputting the identified genes to The Library of Integrated Network-based Cellular Signatures led to many chemopreventive agents of natural origin that produce similar gene expression profiles to that of AM. The mouse sera analysis revealed many immune system-related markers, suggesting the immunosuppressant effect of AM. Finally, a separate study on 5-fluorouracil resistant HCT116 subclones shows the cells possess polyploidy morphology and when the subclones were treated with both AM and 5-fluorouracil, the number of viable cells decreased significantly. The demonstrated effectiveness of AM suggests a potential therapeutic medicine for CRC.
關鍵字(中) ★ 大腸癌
★ 基因晶片
★ 黃耆
★ 異種移植		 關鍵字(英) ★ Colorectal cancer
★ Microarray
★ Astragalus membranaceus
★ xenograft
論文目次 摘要 i
Abstract ii
Acknowledgement iii
Table of Contents iv
List of Figures vi
List of Tables vii
List of Abbreviations viii
Chapter 1. Introduction 1
1.1 Epidemiology: Diagnosis and Staging 1
1.2 Treatment 2
1.3 Molecular Analysis of Colorectal Cancer 3
1.4 Herbal Medicine in the Treatment of Colorectal Cancer 4
1.5 Astragalus membranaceus (Fischer) Bge. var. mongolicus (Bge.) Hsiao (AM) in Cancer Treatment 6
1.6 Significance and Project Aims 7
Chapter 2. Materials and methods 9
2.1 Ethics Statement 9
2.2 Cell Lines and Culture Condition 9
2.3 Preparation of AM 9
2.4 alamarBlue® Viability Assay 10
2.5 Trypan Blue Viability Assay 10
2.6 FACS Analysis 10
2.7 Tumor Xenografts Experiment 11
2.8 Microarray Experiment 11
2.9 Analysis of Microarray Gene Expression Profile 12
2.10 Real-time PCR Validation 13
2.11 Database Analysis for Differentially Expressed microRNAs 13
2.12 Identification of Compound Signature Using LINCS 14
2.13 Western Blotting 14
2.14 Immunohistochemical Staining 15
2.15 Yeast Proteome Microarray Assay 15
2.16 Yeast Proteome Array Data Processing 16
2.17 Statistical Analysis 17
Chapter 3. Effect and Molecular Analysis of AM on HCT116 Cells in vitro and in vivo 18
3.1 Introduction and Objective 18
3.2 Results 20
3.2.1 AM Suppresses The Growth of HCT116 Cells in vitro 20
3.2.2 The Inhibitive Effects of AM in vivo 21
3.2.3 Bioinformatics Analysis of Molecular Mechanisms 21
3.2.4 Focal Analysis on miRNA 23
3.2.5 Experimental Validation of Bioinformatics Analysis on Migration and Angiogenesis of HCT116 cells 24
3.2.6 Drug Predictions Using LINCS 24
3.2.7 Identification of Differentially Expressed Yeast Genes from Mouse Sera 24
3.2.8 Functional Classification, Pathway and Network Analysis 25
3.3 Discussion 27
Chapter 4. Microarray Analysis of 5-fluorouracil-induced Resistant HCT116 Colorectal Cancer Cells 36
4.1 Introduction and Objective 36
4.2 Results 37
4.2.1Effect of 5FU on Proliferative Activity of HCT116 Parental and HCT116 Resistance Subclones 37
4.2.2 Hierarchical Clustering and Functional Analysis of HCT116 Parental and Resistant Subclones 37
4.2.3 Characteristics of HCT116 Resistant Subclones and AM Effect in Combined Therapy 38
4.3 Discussion 38
Chapter 5. Conclusion 40
References 41
Supplementary Table 1. 92
Supplementary Table 2. 93
Supplementary Table 3. 94
Supplementary Table 4. 95
Supplementary Table 5. 96
Supplementary Table 6. 97
Supplementary Table 7. 98
Supplementary Figure 1. 99
Supplementary Figure 2. 100
Supplementary Figure 3. 101
Supplementary Figure 4. 105
Supplementary Figure 5. 109
Supplementary Figure 6. 113
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指導教授 李弘謙、蘇立仁(Hoong-Chien Lee Li-Jen Su) 審核日期 2016-7-18
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