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研究生:邱莉喻
研究生(外文):Chiou, Liyu
論文名稱:探討山竹中之天然化合物garcinol預防小鼠腸癌形成及抑制人類前列腺癌細胞的生長
論文名稱(外文):Studies on chemopreventive potential of garcinol in mouse colon carcinogenesis and inducing growth inhibition in human prostatic carcinoma cells
指導教授:潘敏雄潘敏雄引用關係
指導教授(外文):Pan, Minhsiung
口試委員:陳威仁蔡美玲
口試委員(外文):Chen, WeijenTsai, Meiling
口試日期:2011-07-26
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:水產食品科學研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:152
中文關鍵詞:garcinol異常腺窩結腸炎
外文關鍵詞:garcinolACF (Aberrant crypt foci)DSS (Dextran sulfate sodium)colitis
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garcinol 為polyisoprenylated benzophenone衍生物由山竹中果皮分離而得,目前研究指出具有抗發炎、抗癌症活性以及預防大鼠腸道內異常腺窩的產生。然而,有關garcinol對於腸道發炎形成癌症其機制尚未被探討,在本篇研究目的飲食中補充garcinol對於誘導小鼠腸發炎所形成腸癌之化學預防能力。六週齡之ICR 小鼠腹腔內注射10 mg/kg AOM一星期後,給於小鼠飲用2 % DSS 水一個禮拜,再更換為一般的飲用水,直到25週後犧牲。在25週犧牲後,飼料中添加garcinol (250 ppm and 500 ppm) 有顯著性的降低腫瘤大小及發生率。給於garcinol 500 ppm可以減少經AOM和DSS造成的腸長度縮短的現象。以西方墨點法分析腸道腫瘤中蛋白質的表現,其結果證實garcinol會增加凋亡指標cleavage PARP 以及降低COX-2、VEGF、PCNA和b-catenin的含量。飲食中添加garcinol具有抑制AOM和DSS誘導的腸癌發展,這些證據指出garcinol 可能可作為預防人類腸道潰瘍引起的腸癌化學預防試劑。
Garcinol is the polyisoprenylated benzophenone derivative isolated from fruit rind of Garcina indica tropical region. Previous studies demonstrated garcinol possess anti-inflammatory, anti-cancer activities, and chemopreventive efficacy on colonic aberrant crypt foci in rat. However, the effect and mechanisms of garcinol against colitis-associated colon carcinogenesis is yet to be investigated. In this study, we evaluated chemopreventive effect of dietary garcinol on colitis-induced colon carcinogenesis in mice. Six-week ICR mice were injected i.p. with 10 mg/kg azoxymethane (AOM). After one week, 2 % dextran sodium sulfate (DSS) was administered in the drinking for seven days, and then changes ordinary water for following for twenty-five weeks. Our results shown that feeding garcinol (250 ppm and 500 ppm) for twenty-five weeks significantly reduced the tumor size and incidence in mouse colon. Dietary garcinol (500 ppm) also significantly abated colon length induced by AOM/DSS. In addition, western bolt analysis showed that garcinol markedly increased cleavage of PARP, a marker of apoptosis, as well as down-regulated cyclooxygenase-2 (COX-2), proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF) and b-catenin in AOM/DSS-induced colon carcinogenesis in mice. Our results suggested that garcinol might be as potential a chemopreventive agent for colitis-associated colon cancer.
第壹部分
Chemoprevention of colonic tumorigenesis by dietary garcinol in AOM/DSS-induced carcinogenesis model
中文摘要要 .................................................................................................................. 2
英文摘要 .................................................................................................................. 3
第壹章、緒論 .................................................................................................. 3
第貳章、文獻回顧 .......................................................................................... 5
第一節、大腸直腸癌流行病學概況 ...................................................... 5
2.1.1. 大腸與直腸之構造及生理功能 ........................................... 5
2.1.2. 大腸直腸癌之成因及危險因子 ........................................... 6
2.1.3. 大腸直腸癌臨床症狀 ........................................................... 7
2.1.4. 大腸直腸癌診斷方式 ........................................................... 8
2.1.5. 大腸直腸癌治療方式 ........................................................... 9
第二節、大腸直腸癌之形成 ................................................................ 10
2.2.1. Aberrant crypt foci(ACF)與大腸直腸癌關係 ...................... 11
2.2.2. 大腸直腸癌相關基因之表現 ............................................. 13
第三節、發炎與大腸直腸癌形成 ........................................................ 16
第四節、誘導大腸直腸癌之動物模式 ................................................ 20
第五節、Garcinol簡介 ......................................................................... 24
第参章、研究目的與實驗架構 ............................................................ 27
第肆章、實驗材料與方法 ............................................................................ 28
第一節、實驗材料 ................................................................................ 28
4.1.1. 儀器與廠牌 ......................................................................... 28
4.1.2. 藥品來源及試劑 ................................................................. 29
第二節、實驗方法 ................................................................................ 32
4.2.1. DSS及AOM/DSS誘發小鼠腸道致癌實驗分組及設計 .. 32
4.2.2. 小鼠組織樣品的收集(血液、肝臟、腎臟、脾臟及結腸)34
4.2.3. 結腸中ACF (aberrant crypts foci) 和腫瘤數目及分布情形 ........................................................................................................ 35
4.2.4. 蛋白質萃取與西方墨點法 ................................................. 35
4.2.5. 組織染色(Histochemistry) .................................................. 39
4.2.6. 統計分析 ............................................................................. 42
第伍章、結果與討論 .................................................................................... 43
第一節、DSS及AOM/DSS誘發ICR小鼠腸道致癌過程體重及器官的變化 .................................................................................................... 43
第二節、Garcinol對ICR小鼠腸道ACF生成及病理變化特徵 ....... 43
第三節、AOM/DSS誘發ICR小鼠腸道腫瘤大小變化之情形 ......... 44
第四節、Garcinol具抑制DSS及DSS/AOM所誘發ICR小鼠發炎反
應 ............................................................................................................ 44
第五節、Garcinol具抑制DSS/AOM所誘發ICR小鼠腸道細胞增生及誘導凋亡 ................................................................................................ 44
第六章、結論 ................................................................................................ 46
第貳部分
The inhibition of PC-3 cells growth by garcinol in xenograft tumor model
中文摘要 ................................................................................................................ 58
英文摘要 ................................................................................................................ 59
第壹章、緒論 ................................................................................................ 60
第貳章、文獻回顧 ........................................................................................ 61
第一節、癌症 (Cancer) ........................................................................ 61
2.1.1. 癌症概述 ............................................................................. 61
2.1.2. 前列腺癌流行病學概況 ..................................................... 62
2.1.3. 前列腺構造及生理功能 ..................................................... 64
2.1.4. 前列腺癌形成之過程 ......................................................... 65
2.1.5. 前列腺危險因子 ................................................................. 67
2.1.6. 前列腺癌診斷方式 ............................................................. 72
2.1.7. 前列腺癌臨床病理分期 ..................................................... 75
2.1.8. 前列腺癌治療方式 ............................................................. 76
第二節、細胞凋亡 (Apoptosis) ........................................................... 79
2.2.1. 細胞凋亡介紹 ..................................................................... 79
2.2.2. 細胞凋亡之型態 ................................................................. 81
2.2.3. 細胞凋亡之機制 ................................................................. 83
2.2.4. 調節凋亡相關蛋白質(Regulation of the apoptotic proteins) ........................................................................................................ 85
第三節、自體吞噬 (Autophagy) .......................................................... 89
2.3.1. 自體吞噬介紹 (Autophagy) ............................................... 89
2.3.3. 自體吞噬與癌症 ................................................................. 96
2.3.4. 在癌症自體吞噬訊息傳遞路徑 ......................................... 97
2.3.5. 自體吞噬抑制劑 ................................................................. 99
第四節、自體吞噬與凋亡相關性 ................................................ 99
第参章、研究目的與實驗架構 .................................................................. 101
第肆章、實驗材料與方法 .......................................................................... 102
第一節、實驗材料 .............................................................................. 102
4.1.1. 儀器與廠牌 ....................................................................... 102
4.1.2. 藥品來源及試劑 ............................................................... 103
第二節、實驗方法 .............................................................................. 106
4.2.1. 黏著性癌細胞解凍與培養 ............................................... 106
4.2.2. 細胞存活率試驗 ............................................................... 107
4.2.3. 染色體DNA之片段化 .................................................... 108
4.2.4. 流式細胞分析儀分析 ....................................................... 109
4.2.5. 自體吞噬細胞死亡之比率分析 ........................................ 110
4.2.6. 細胞內粒腺體膜電位之分析 ............................................ 112
4.2.7. 前列腺癌細胞異種移植分析實驗設計 ............................ 113
4.2.8. 前列腺癌細胞異種移植之Caspase 活性測定 ................ 114
4.2.9. 前列腺癌細胞異種移植之蛋白質電泳與西方墨點法分析 ....................................................................................................... 115
4.2.10. 統計分析 .......................................................................... 115
第伍章、結果與討論 ................................................................................... 116
第一節、以不同濃度之garcinol對人類前列腺癌細胞生長之影響 116
第二節、Garcinol具有導致前列腺癌細胞株PC-3之DNA片段化情形及細胞內粒腺體膜電位之分析 ....................................... 116
第三節、Garcinol能趨於PC-3細胞誘導自體吞噬之情形 .............. 117
第四節、Garcinol對於異種移植 (xenograft) 於動物實驗之影響 .. 118
第六章、結論 .............................................................................................. 120
第七章、參考文獻 ...................................................................................... 131
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