臺灣博碩士論文加值系統

口腔癌是世界上十大最常見的癌症之一，每年有超過50萬人確診；於台灣十大癌症死因排名第四，口腔癌的根治相當困難，復發率高且復發時間短，最近研究顯示，癌症的治癒與其轉移有關。上皮-間質細胞轉換(Epithelial-Mesenchymal Transition)是在癌細胞的轉移中扮演著很重要的機轉，上皮細胞失去細胞間的黏附性，進而轉換成具有高度轉移能力的間質細胞。調節EMT的分子機制有許多種，Bmi1(the polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog)蛋白就是其中之一。過去研究指出，Bmi1具有許多功能，包含調節細胞生長、調節EMT發生及幹細胞的自我更新…等。在本篇研究中，主要目的為找到可以抑制Bmi1蛋白表現的天然化合物。我們發現，大黃根部主要成分大黃素，可以降低OECM-1及SAS細胞中的Bmi1蛋白表現。另外，大黃素能夠抑制這兩株細胞的增生、改變細胞型態、降低爬行以及侵襲能力。此外，大黃素能透過抑制Bmi1蛋白下游AKT路徑抑制EMT。總體上，我們的研究證明了大黃素對於口腔癌的治療可能有相當的潛力。

Oral cancer is a malignant neoplasia which arises on the lip or oral cavity. Oral cancer is frequently associated with metastasis to draining lymph nodes, and this event is correlated with poor prognosis and reduced survival rates. Epithelial-mesenchymal transition (EMT) is a crucial event required for the invasion and progression of carcinogenesis, in the progress of EMT, the epithelial cells lose cell-cell adhesion to turn into mesenchymal cells and gain migration ability. There are many pathways and transcription factors can regulate EMT, Bmi1 was one of them. Bmi1, the polycomb group protein B lymphoma Mo-MLV insertion region 1 homolog, induces cell cycle, regulates EMT transition, maintains the self-renewal capacity of stem cells, and is frequently overexpressed in human cancers, including oral cancer. In this study, we aim to test whether natural compounds could inhibit the expression of Bmi1. We found that emodin, a predominant compound from the root of rhubarb can decrease Bmi1 expression in OECM-1 and SAS cells. Moreover, emodin inhibit cell proliforlation and changed cell morphology in these two cells. Besides, emodin could inhibit migration and invasion ability. Emodin also inhibited EMT though down-regulating Akt pathway. Overall, our study indicated that emodin have the potent therapeutic effect in oral cancer.

第一章 研究源起 1
第一節 惡性腫瘤與轉移 1
1.2.1 細胞轉形及起始階段(Transformation and initiation): 2
1.2.2 腫瘤生長階段(Neoplastic growth): 3
1.2.3 血管新生階段 4
1.2.4 癌細胞侵犯階段(Invasion): 5
1.2.4.1 細胞的延展作用(Extension of directed protrusion) 6
1.2.4.2 細胞的錨定作用(Anchoring of protruisions to the substrate or the ECM)： 6
1.2.4.3 Actomyosin filament 的鏈結階段： 7
1.2.4.4 最終階段： 7
第二節 口腔癌 9
2.1吸菸 9
2.2飲酒 10
2.3 嚼檳榔 10
2.4其他 11
第二章 文獻回顧 14
第一節 上皮細胞-間質細胞轉換 (Epithelial-mesenchymal transition; EMT) 14
2.1.1 第一階段 侵犯 (invasion) 15
2.1.2 第二階段 內滲 (Intravasation) 15
2.1.3 第三階段 外滲 (Extravasation) 15
第二節Bmi1 18
第三節 大黃素 19
第四節 研究動機 20
第五節 實驗設計 21
第四章 研究結果 22
第一節 天然化合物在口腔癌中抑制Bmi1的效果 22
第二節 大黃素對於OECM-1及SAS細胞存活率影響 23
第三節 大黃素在OECM-1及SAS細胞中抑制Bmi1的效果 25
第四節 大黃素對於OECM-1及SAS細胞型態的改變 26
第五節 大黃素在OECM-1及SAS中對於細胞爬行能力的影響 27
第六節 大黃素對於OECM-1及SAS細胞中侵襲能力的影響 29
第七節 大黃素對於OECM-1及SAS細胞中EMT相關的生物標誌影響 31
第八節、大黃素對於OECM-1和SAS細胞中AKT/ β-catenin/GSK-3β/Aurora A路徑的影響 33
第五章 討論 36
第六章 結論 39
第七章、材料與方法 40
第一節 研究材料 40
7.1.1 、細胞株 40
7.1.2 、藥品與試劑 40
7.1.3 抗體 42
7.1.4主要儀器與耗材 42
第二節 細胞培養 (Cell culture) 44
7.2.1 細胞培養與繼代 44
7.2.2 細胞冷凍保存 44
7.2.3 活化冷凍細胞 45
第三節 細胞型態觀察 (Cell morphology observation) 45
第四節 細胞增生速率分析 (Cell proliferation assay) 45
第五節 細胞存活率分析 (MTT assay) 46
第六節 西方墨點法 (Western blot) 47
7.6.1 萃取蛋白質 (Protein extraction) 47
7.6.2 蛋白質濃度測定 47
7.6.3 蛋白質電泳分析 (SDS-PAGE) 48
7.6.4 轉漬與影像呈現 49
第七節 細胞爬行能力試驗 (Wound Healing Assay) 50
第八節 細胞侵襲實驗 (Invasion assay) 51
第九節 統計方法 52
第八章 參考文獻 53

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