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研究生:何秉穎
研究生(外文):Bing-Ying Ho
論文名稱:紅麴發酵產物在癌症預防與治療中所扮演的角色
論文名稱(外文):The roles of Monascus-fermented products on cancer chemopreventive and therapeutic modulation
指導教授:潘子明潘子明引用關係
指導教授(外文):Tzu-Ming Pan
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:225
中文關鍵詞:紅麴轉移血管新生化學預防
外文關鍵詞:MonascusMetastasisAngiogenesisChemoprevention
相關次數:
  • 被引用被引用:1
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  • 下載下載:112
  • 收藏至我的研究室書目清單書目收藏:3
紅麴發酵產物為深具東方特色之傳統食品,其二次代謝產物經科學研究證實具有抑制腫瘤轉移、抗發炎、降低膽固醇生成等多項作用。因此,在學理上十分適合運用於抑制腫瘤治療過程中可能引發的腫瘤轉移及組織發炎反應的輔助性治療。
本論文第一部份利用小鼠肺癌細胞 (Lewis lung carcinoma; LLC) 植入C57/BL6 小鼠所形成 LLC 荷瘤小鼠,探討給與紅麴米餵食所產生之效應。植入 5 x 105 LLC 細胞之小鼠,同時餵食 2% 紅麴米,經 14 天後與未攝食紅麴米組比較腫瘤大小,紅麴米抑制達 51.1% 之多。以紅麴乙醇萃出物 (red mold rice ethanol extract; RMRE) 進行體外侵襲與轉移試驗顯示,RMRE 可抑制透過介白質六號 (Interleukin-6; IL-6) 所活化,與侵襲及轉移能力增加相關的基質金屬蛋白酶 (matrix metalloproteinases; MMPs),包括 MMP-2 與 MMP-9。進一步在 RMRE 合併臨床化療藥物治療的效力上發現,RMRE 不僅造成 LLC 細胞的細胞週期停止於 G1 期,同時隨著劑量提升而增加誘發其細胞凋亡現象。
第二部份探討 RMRE 抑制 LLC 荷瘤小鼠血清中血管內皮生長因子 (vascular endothelial growth factor; VEGF) 上升,同時降低荷瘤小鼠轉移的發生。結果指出 RMRE 中的 monacolin K (MK) 可能扮演著關鍵性角色,包括利用體外轉移與血管新生試驗證實 MK 抑制 LLC 細胞的轉移侵襲能力,同時經反轉錄聚合酶鏈反應 (reverse transcription polymerase chain reaction; RT-PCR) 得知,MK 藉由降解 VEGF 基因表現而抑制 LLC 細胞分泌 VEGF 誘發內皮細胞血管之生成。
第三部份以雞胚胎尿囊絨毛膜 (chorioallantoic membrane; CAM) 試驗探討 RMRE 與紅麴抗氧化物 dimerumic acid (DMA) 對於腫瘤血管新生與細胞內滲之作用。RMRE 與 DMA 隨著劑量與時間增加對人類大腸癌細胞株,SW480 與 SW620 細胞,具有劑量關係。同時經由分析雞胚 CAM 下方所含有人類 Alu 序列片段結果顯示,RMRE 可抑制於 Matrigel 包覆有 SW620 細胞之植體從雞胚 CAM 上方自發性內滲轉移至 CAM 下方的能力。含 SW620 細胞之植體於 CAM 中可增加 75.3±11.6% 之新生血管生成,然 RMRE 可隨劑量增加而顯著抑制 SW620 所誘發之 CAM 血管新生。最後經由 RT-PCR、西方墨點轉漬分析與酪蛋白酶譜分析顯示 RMRE 藉由抑制 MMP-7 活性達到降低腫瘤轉移發生。
以上顯示,利用 LLC 荷瘤小鼠或雞胚胎 CAM 等動物模式皆證實以膳食補充紅麴米方式可達到抑制腫瘤生長與轉移之發生。因此,包括 RMRE、MK、AK 與 DMA 於未來相當具潛力研究發展,使成為一無毒性、天然之化學預防膳食補充品,或作為癌症治療輔助劑。

The Monascus fermented products are of special interest because of their use in the production of a range of oriental fermented foods. The secondary metabolites of Monascus in the modern research were confirmed had the suppression tumor metastasis, anti-inflammation, the suppression cholesterol production and so on many functions. Therefore, Monascus metabolites extremely suit to utilize cancer therapy to suppress the tumor metastasis and the inflammation responded.
The first section described the effects of red mold rice on Lewis lung carcinoma (LLC) bearing in syngeneic C57BL/6 mice. Oral administration of 2% red mold rice and implant 5 × 105 LLC cells in mice for 14 days significantly inhibited the tumor progress (51.1%). In vitro assays, the investigation involved the effects of red mold rice ethanol extract (RMRE) on LLC cells invasion and metastasis. RMRE interference on Interleukin-6 (IL-6) induced cell invasion and metastasis gene expressions of matrix metalloproteinases (MMPs) contained MMP-2 and MMP-9. Moreover, the assessment of chemotherapeutic efficiency combined clinical drugs used on tumor cell viabilities have showed that RMRE not only significantly arrested LLC cells G1 phase, but also induced cell apoptosis following arise the doses of RMRE treatment.
The secondary part represented that oral administration with RMRE or dramatically inhibited the metastatic ability of LLC-bearing mice caused by the decline of serum vascular endothelial growth factor (VEGF) levels compared with untreated metastatic groups. These results indicated that MK was a key antimetastatic and antiangiogenesis compound in RMRE and proved that by Matrigel-coating transwell and tube-forming assays, as well as reverse transcription polymerase chain reaction (RT-PCR) illustrated the downregulation of VEGF-stimulated invasive activity in LLC cells.
The third section showed RMRE and DMA significantly inhibited the proliferation of SW480 and SW620 human colorectal carcinoma cells in dose- and time-dependent manner. Moreover, SW620 cells could growth in Matrigel grafts and spontaneously intravasation from the upper to the lower of chick embryo chorioallantoic membrane (CAM) model, which detected human Alu genomic DNA by PCR amplification from the lower CAMs at RMRE-untreated group. The percentage of neovascularization was increased 75.3±11.6% by SW620 cells onplant with Matrigel grafts on the CAM model. However, addition of RMRE significantly reduced CAM neovascularization in dose-dependent effect. Finally, we have finding that RMRE and DMA effectively decreased activity of MMP-7 determined by RT-PCR, Western blotting, and casein zymography assays.
In summary, using the food-based approach to cancer prevention has shown that Monascus-fermented products ingestion inhibited both the primary tumor growth and metastatic progression on both LLC-bearing mice and chick embryo CAM models in vivo. Therefore, application of RMRE, MK, AK and DMA may serve as a non-toxic natural chemopreventive or antineoplastic agent for further development of cancer adjuvant chemotherapy. We conclude that Monascus is a highly potential herb for cancer therapy especially in tumor chemotherapy, the more appropriate application worthy to be studied in the future.

目錄
表目錄 vi
圖目錄 vii
縮寫表 ix
中文摘要 xiv
Abstract xvi
第一章 文獻回顧 1
第一節 紅麴菌的發現 1
第二節 紅麴的製作與使用 3
一、 古籍文獻記載 3
二、 紅麴發酵培養與生產方法 4
(一) 傳統民間方法 4
(二) 實驗室液態培養法 4
(三) 實驗室固態培養法 5
(四) 工業化生產與應用 7
(五) 不同基質取代與最適化生產 8
第三節 紅麴之二次代謝產物 9
一、 Monacolins 10
二、 紅麴色素 12
三、 橘黴素 (citrinin) 18
四、 γ-胺基丁酸 (γ-aminobutyric acid; GABA) 19
五、 Dimerumic acid 20
第四節 紅麴發酵產物之生理活性作用 21
一、 調節血脂與抑制膽固醇生合成 21
二、 調節血糖與抑制脂肪生成 25
三、 調節血壓與心血管疾病預防 31
四、 抗氧化與延緩衰老 33
五、 器官毒副作用與過敏性反應 39
六、 癌症預防與腫瘤毒殺作用 42
第五節 食品輔助治療與癌症化學預防應用 46
一、 癌症成因與癌症化學預防 47
二、 惡性腫瘤特徵 52
(一) 自給自足之生長訊號 (Self-sufficiency in growth signals) 52
(二) 對抑制生長的訊號不反應 (Insensitive to anti-growth signals) 54
(三) 逃避細胞凋亡機制 (Evading apoptosis) 54
(四) 不受限之複製能力 (Limitless replicative potential) 54
(五) 維持血管新生 (Sustained angiogenesis) 55
(六) 侵入組織及轉移 (Tissue invasion and metastasis) 56
(七) 微環境之發炎反應 (Inflammatory microenvironment) 56
三、 天然活性化合物與腫瘤分子標靶 59
四、 中醫藥輔助與替代性醫療 59
第二章 研究動機與目的 64
第一節 研究動機 64
一、 保健預防的觀念提升 64
二、 飲食防癌是最新趨勢 65
三、 紅麴應用開發 66
第二節 研究目的 67
一、 探討紅麴發酵多樣性功能成分輔助癌症治療之可行性 67
二、 紅麴發酵產物調控之分子機轉探討 67
第三章 紅麴萃出物合併化療或放療之生物效應 68
第一節 前言 68
第二節 研究材料與方法 69
一、 儀器設備 69
二、 藥品試劑與耗材 69
三、 細胞培養 70
四、 細胞生長抑制試驗 72
(一) MTT 分析法 72
(二) 結晶紫染色分析法 72
五、 細胞週期與細胞凋亡分析 73
(一) 流式細胞儀分析法 73
(二) 細胞核螢光染色法 74
六、 體外細胞移行與侵襲能力試驗 75
(一) 腫瘤細胞移動能力測試 (In vitro wound healing assay) 75
(二) 腫瘤細胞侵襲能力測試 (Cell invasive assay) 75
七、 反轉錄-聚合酶鏈反應 (RT-PCR) 分析基因表現 75
(一) 總核醣核酸萃取 75
(二) 反轉錄 cDNA 合成 77
(三) 聚合酶鏈反應 (PCR) 77
八、 小鼠荷瘤模式試驗 78
九、 統計分析與製圖 81
第三節 研究結果 81
一、 建立腫瘤細胞株之紅麴萃出物半抑制劑量 81
二、 RMRE 抑制LLC 小鼠肺癌細胞株生長 83
三、 RMRE 與化療藥物對 LLC 小鼠細胞株之細胞凋亡作用 84
(一) 化療藥物誘導腫瘤細胞凋亡 84
(二) DOX 併用 RMRE 提高細胞毒殺率 87
四、 細胞轉移相關因子分析 90
(一) RMRE 降低 LLC 腫瘤細胞移行與侵襲能力 90
(二) 細胞轉移基因變化分析 92
五、 小鼠移植 LLC 腫瘤試驗 (LLC-bearing mice model) 94
(一) 紅麴米餵食比例與小鼠體重關係比較 94
(二) 添加 2% 紅麴米飼料延緩 LLC 腫瘤形成 96
(三) 荷瘤小鼠餵食 RMR 提昇 CDDP 化療效果 99
(四) 放射線治療合併紅麴之輔助效應 99
第四節 結論 104
第四章 紅麴二次代謝物對肺腫瘤進展與癌轉移之抑制作用 105
第一節 前言 105
第二節 研究材料與方法 108
一、 RMRE 配製與分析 108
二、 小鼠細胞株培養 108
三、 腫瘤細胞移行與侵襲能力 108
四、 體外毛細管形成試驗 (In vitro capillary tube formation assay) 111
五、 流式細胞儀分析 111
六、 LLC 小鼠荷瘤模式 (LLC-bearing mice model) 113
七、 組織切片染色 (Hematoxylin and Eosin; H&E) 113
八、 酵素免疫化學分析 (ELISA) 114
九、 統計分析與製圖 114
第三節 紅麴對於肺腫瘤進展與癌轉移之研究結果 114
一、 RMRE抑制腫瘤生長能力優於單一成分 MK 114
二、 荷瘤動物之腫瘤重量與肺轉移比較 116
三、 Ankaflavin 參與腫瘤細胞生長抑制之加乘作用 116
四、 RMRE 所含 MK 減少自發性腫瘤轉移 119
五、 紅麴二次代謝物影響 VEGF 表現 119
六、 MK 調控 LLC 細胞株之VEGF 表現 124
七、 MK 抑制血管生成 124
第四節 結論 129
第五章 探討紅麴抑制大腸直腸癌轉移與血管新生作用機轉 130
第一節 前言 130
第二節 研究材料與方法 131
一、 生長抑制分析 (MTT) 131
二、 雞胚胎轉移試驗 (Chick embryo metastasis assay) 131
三、 Matrigel 包覆細胞之 CAM分析 (Matrigel encapsulated CAM assay) 133
四、 RT-PCR 基因表現分析 133
(一) 總核醣核酸萃取 133
(二) 反轉錄 cDNA 合成 134
(三) 聚合酶鏈反應 (PCR) 134
五、 蛋白質分析 136
(一) 蛋白質萃取與定量 136
(二) SDS聚丙烯酰胺膠凝體電泳法 (SDS-PAGE) 136
(三) 西方墨點轉漬分析法 (Western blotting) 137
六、 酪蛋白酶譜分析 (Casein zymography) 137
第三節 紅麴抑制大腸直腸癌細胞株轉移與血管新生之研究結果 138
一、 RMRE 對於細胞存活率具有時間與劑量效應 138
二、 RMRE 抑制腫瘤衍生之體外血管新生 138
三、 體內雞胚胎 CAM 模式證實紅麴抑制腫瘤轉移與血管新生 141
(一) RMRE 抑制 SW620 細胞之內滲作用 141
(二) RMRE 抑制腫瘤誘導之血管新生作用 141
第四節 紅麴抑制 MMP-7 表現與癌侵襲作用機轉之研究結果 143
一、 RMRE 抑制MMP-7 表現降低 SW620 細胞之侵襲能力 143
二、 H2O2 與 DMA 對 SW620 大腸癌細胞株生長之影響 143
三、 紅麴抗氧化物 DMA 抑制 H2O2 誘導之侵襲作用 147
四、 DMA 抑制 H2O2 所誘發的 MMP-7 表現 147
五、 紅麴抗氧化物 DMA 抑制 H2O2 誘導之 MAPK 之活化 150
六、 H2O2 透過 JNK/c-Jun 與 ERK/c-Fos 活化轉錄因子啟動 MMP-7 表現 155
第五節 結論 158
第六章 總結 161
第一節 紅麴具輔助改善化學治療與放射線治療副作用之潛力 161
第二節 紅麴降血脂二次代謝物 MK 抑制腫瘤轉移 163
第三節 RMRE 抑制大腸直腸癌轉移與血管新生作用 164
第四節 紅麴抗氧化物 DMA 抑制H2O2 所誘發之 MMP-7 表現 164
第七章 參考文獻 165
第八章 附錄 193
附錄一、博士班就讀期間研究成果 193
一、 出席國內研討會壁報展出 193
二、 出席國際會議與壁報展出 194
三、 出席國際會議與英文口頭報告 194
四、 學術論文 (SCI) 195
五、 得獎獎項 196
附錄二、代表著作全文 197
附錄三、獎狀 216
附錄四、自述 225

表目錄

表 1-1 世界衛生組織訂定之身體質量指數與體重區分標準 26
表 1-2 台灣成人肥胖定義 28
表 3-1 本研究包含之細胞株種類 71
表 3-2 PCR 分析引子 79
表 3-3 荷瘤 (LLC) 小鼠模式之化學預防與輔助治療組別 80
表 3-4 紅麴發酵萃出物濃度對不同細胞生長抑制率比較 82
表 3-5 根據體表面積換算動物劑量等效之人體劑量 95
表 4-1 RMRE 中 MK 與 AK 之濃度比較 109
表 4-2 RMRE 與 MK 對 LLC 荷瘤鼠腫瘤重量與肺轉移之影響 117
表 4-3 種植 1 × 106 LLC 細胞之荷瘤小鼠肺轉移數量 120
表 5-1 雞胚胎 CAM 試驗與人類 MMP-7 之引子序列 135
表 6-1 紅麴發酵產物生物活性之研究進展 162


圖目錄

圖 1-1 紅麴菌生活史 2
圖 1-2 紅麴米固態發酵製程 6
圖 1-3 Monacolin K 的相關結構化合物 11
圖 1-4 Monacolin K 前驅物與其生合成過程 13
圖 1-5 MK 包含四個可與 HMG-CoA reductase 結合作用區位 14
圖 1-6 Monascin 黃色素生合成路徑 16
圖 1-7 Dimerumic acid 氧化形成氧化胺自由基 22
圖 1-8 胰島素的能量調節與肥胖及第二型糖尿病形成之間的關係 29
圖 1-9 肥胖引起之高血壓,其胰島素與血壓間之關聯性假說 32
圖 1-10 圖解 Statins 多樣性的生物功效 37
圖 1-11 Statins 引發肌病變可能的因素 41
圖 1-12 典型植物化學預防物質及其來源 48
圖 1-13 癌化過程與化學預防機制 50
圖 1-14 增加癌症風險的可能因子 51
圖 1-15 惡性腫瘤標誌 53
圖 1-16 急性發炎與慢性發炎誘發癌化之關聯性對照 58
圖 1-17 食品成分物質預防與治療癌症之分子目標 60
圖 3-1 體外細胞侵襲試驗圖解 76
圖 3-2 LLC 細胞生長及抑制之外觀及聚落分析 85
圖 3-3 LLC 的細胞週期分析 86
圖 3-4 RMRE 與化療藥物對 LLC 細胞的作用 88
圖 3-5 DOX 合併 RMRE 促進 LLC 細胞毒殺作用 89
圖 3-6 LLC 細胞移行及侵襲能力測試 91
圖 3-7 LLC 細胞的 MMPs mRNA 表現 93
圖 3-8 添加不同比例紅麴米粉末餵食之 C57BL/6 小鼠體重變化圖 97
圖 3-9 注入 LLC 細胞量與腫瘤增長大小比較 98
圖 3-10 C57BL/6 小鼠化療存活率與腫瘤形成體積 100
圖 3-11 放射線治療組之試驗結果 103
圖 4-1 各種抑制肺癌形成之抑制劑結構 106
圖 4-2 以氫核子共振光譜鑑定 RMRE 中的 AK 110
圖 4-3 藉由不同輔助治療改變原位腫瘤大小 115
圖 4-4 MK 與AK 合併抑制細胞生長並促進細胞凋亡 118
圖 4-5 LLC 荷瘤小鼠肺轉移之組織染色分析 121
圖 4-6 RMRE 與 MK 對於組織血清 VEGF 表現的影響 123
圖 4-7 MK 抑制 VEGF 誘導 LLC 行侵襲作用穿過 Matrigel coating 膜 125
圖 4-8 MK 抑制 LLC 誘導的 SVEC 4-10 內皮細胞血管形成 127
圖 5-1 雞胚胎轉移模式 132
圖 5-2 RMRE 抑制大腸癌細胞生長具劑量與時間關係 139
圖 5-3 RMRE 影響人類內皮細胞生長與血管生成作用 140
圖 5-4 以雞胚胎植入模式分析腫瘤內滲作用 142
圖 5-5 RMRE 抑制雞胚尿囊絨毛膜血管新生 144
圖 5-6 RMRE 藉由降解 MMP-7 表現以抑制 VEGF 調控之細胞侵襲作用 145
圖 5-7 DMA 與 H2O2 對於SW620 大腸癌細胞株存活率之影響 148
圖 5-8 DMA 預防外添加 H2O2 引起之 SW620 大腸癌細胞株侵襲作用 149
圖 5-9 H2O2 對於SW620 大腸癌細胞株 MMP-7 表現與活化之作用 151
圖 5-10 分析 H2O2 活化 SW620 大腸癌細胞株不同 MAPK 路徑表現 153
圖 5-11 H2O2 對於SW620 大腸癌細胞株 MMP-7 轉錄與活化之調控 156
圖 5-12 圖解 DMA 抑制癌細胞侵襲作用之可能訊息傳遞路徑 159

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