臺灣博碩士論文加值系統

摘要
由於國人腫瘤發生率日益增高, 因此近幾年來研究的方向主要著眼於飲食成份與抑制腫瘤形成之間的相關性。 本實驗主要以小老鼠肝癌細胞株( Hepa1 )及人類肝癌細胞株( HepG2 )為實驗模式, 探討十字花科蔬菜中衍生物indole-3-carbinol (I3C)及indolo[3,2-b]carbinol (ICZ)對於調節解毒酵素QR活性所扮演的角色以及調節之機轉。 結果顯示ICZ之前驅物質I3C在兩種肝癌細胞中皆不具有誘導QR之效果； 反之, ICZ具有誘導QR酵素活性之作用,且誘導能力隨ICZ劑量增加而增加, 同時Hepa1細胞之表現較HepG2細胞敏感； 時間效應之結果顯示細胞於添加ICZ 48小時後有最大的誘導活性; RT-PCR結果指出ICZ誘導QR酵素活性之機轉乃經由增加QR酵素之轉錄作用, 經由QR mRNA含量之增加而達到促進QR酵素之表現的效果。 總言之, I3C對於QR活性於兩種細胞株中皆不具誘導作用, 反之I3C之酸性產物ICZ則具有調節QR酵素活性之作用。 除此之外, 於兩種肝癌細胞中以Hepa1細胞為研究Indole誘導QR機制之較佳之實驗模型。

Abstract
The indoles, such as indole-3-carbinol (I3C) and indolo[3,2-b]carbazole (ICZ), are thought to be the bioactive components in cruciferous vegetables. The induction of detoxification enzymes, such as quinone reductase ( QR ), is closely associated with the chemopreventive effect of various phytochemicals. The present study was aimed to study the role of I3C and ICZ on the regulation of QR in both murine (Hepa1) and human (HepG2) hepatoma cells. The results showed that ICZ is a potent QR inducer, but its parent compound, I3C, had no significant effect on the induction of QR activity. Dose-response experiments indicated that QR activity increased with increasing concentrations of ICZ in both cell lines, and the Hepa1 cells had a higher response than the HepG2 cells. RT-PCR analyses showed that the increased QR activity was due to the increased expression of QR mRNA. In conclusion, ICZ, the acidic polymer of I3C, may increase the expression of QR mRNA, and which then cause the increase of the QR activity. Besides, the Hepa1 cells seems to be a better model to study the ICZ induced QR expression than the HepG2 cells.

目 錄
頁數
中文摘要 Ⅰ
英文摘要 Ⅱ
致謝 Ⅲ
目錄 Ⅴ
表目次 Ⅷ
圖目次 Ⅸ
第一章 緒論 1
第二章 文獻回顧 3
第一節 解毒酵素系統 3
第二節 還原之簡介 6
第三節 十字花科蔬菜之簡介 9
第四節 硫代配糖體之結構及水解作用 10
第五節 indole-3- carbinol ( I3C )之結構及特性 15
第六節 Indolo [ 3,2-b ] carbazole ( ICZ )之結構及特性18
第三章 材料及方法 21
第一節 試劑之選用 21
第二節 儀器之介紹 25
第三節 細胞培養 26
第四節 QR酵素活性之分析 29
第五節 細胞總蛋白質含量測定 32
第六節 細胞中總RNA之萃取 33
第七節 RNA電泳之測定 35
第八節 RT- PCR之測定 38
第九節 DNA電泳之測定 40
第十節 統計分析 41
第四章 結果 42
第一節 I3C對肝癌細胞中QR酵素活性之影響 42
第二節 ICZ對肝癌細胞中QR酵素活性之影響 45
第三節 RNA電泳分析 51
第四節 RT-PCR 54
第五章 討論 58
第六章 結論 63
參考文獻 64
附錄一 Solutions of cell culture 70
附錄二 Preparation of DMEM 72
附錄三 Protocol of cell culture 73
附錄四 Solutions of Quinone reductase enzyme assay 74
附錄五 Protocol of Quinone reductase enzyme assay 77
附錄六 Solutions of crystal violet 79
附錄七 Solutions of cellular RNA isolation 80
附錄八 Protocol of RNA isolation 82
附錄九 Reagents of RNA electrophoresis 84
附錄十 Protocol of RNA electrophoresis 86
附錄十一 Protocol of RT reaction 88
附錄十二 Protocol of PCR reaction 89
附錄十三 Solutions of DNA electrophoresis 90
附錄十四 Protocol of DNA electrophoresis 91
表目次
表一. 解毒酵素之種類 5
表二. 十字花科蔬菜中硫代配糖體之種類 12
表三. 各類十字花科蔬菜中硫代配糖體之含量 14
表四. Primers之序列 38
圖目次
圖一. QR酵素之催化反應 8
圖二. 硫代配糖體之結構 11
圖三. 硫代配糖體之水解 13
圖四. I3C之結構圖 16
圖五. I3C之酸化產物 17
圖六. Indolo [ 3,2-b ] carbazole之結構 20
圖七. 實驗架構圖 28
圖八. QR酵素反應之原理 31
圖九. RT-PCR之原理 39
圖十. I3C於不同時間點對小老鼠肝癌細胞中QR酵素活性 43
的影響
圖十一. I3C於不同時間點對人類肝癌細胞中QR酵素活性 44
的影響
圖十二. 不同濃度之ICZ對小老鼠肝癌細胞中QR酵素活性 47
的影響
圖十三. 不同濃度之ICZ對人類肝癌細胞中QR酵素活性 48
的影響
圖十四. ICZ於不同時間點對小老鼠肝癌細胞中QR酵素活性 49
的影響
圖十五. ICZ於不同時間點對人類肝癌細胞中QR酵素活性 50
的影響
圖十六.不同濃度劑量之ICZ經24小時培養後小老鼠肝癌 52
細胞中RNA之定量結果
圖十七.不同濃度劑量之ICZ經24小時培養後人類肝癌細胞中 53
RNA之定量結果
圖十八. ICZ對於小老鼠肝癌細胞QR mRNA表現之影響 56
圖十九. ICZ對於人類肝癌細胞QR mRNA表現之影響 57

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