臺灣博碩士論文加值系統

在台灣約有超過兩百萬的人口有嚼食檳榔的習慣，而這種行為已經確定會直接造成口腔黏膜下纖維化 (Oral submucous fibrosis, OSF)並和口腔癌有密切的關係，但是對於這類口腔疾病的病理機制以及檳榔子內的生物鹼-檳榔鹼是如何促使OSF的產生，仍然尚未完全了解。肌纖維母細胞 (Myofibroblasts)是一種具有表現平滑肌肌動蛋白 (Alpha smooth muscle actin, α-SMA)標記蛋白質的細胞群，通常會在口腔組織纖維化的過程中分泌膠原蛋白 (Collagen)，並誘導組織細胞間質內的收縮。在本論文中，我們希望探討檳榔鹼刺激頰黏膜口腔纖維母細胞 (Buccal mucosa fibroblasts, BMFs)分化為肌纖維母細胞的分子機制。首先我們確定檳榔鹼對於正常口腔纖維母細胞的生長影響，在不影響細胞存活的濃度下，觀察到當檳榔鹼濃度為10-20μg/ml時，甚至能夠造成細胞增生，並且誘導α-SMA和間質細胞 (Mesenchymal cell)的標記蛋白 Vimentin的表現量上升，為了解由檳榔鹼誘導的α-SMA蛋白質表現機制為何，我們把目標專注在調控上皮-間質細胞轉換過程 (Epithelial-mesenchymal transition, EMT)的轉錄因子上。透過西方墨點法 (Western blot)的結果，發現檳榔鹼誘導頰黏膜纖維母細胞中α-SMA表現量上升時， EMT轉錄因子Zinc finger E-box binding homeobox 1 (ZEB-1)也隨著上升。而在膠原蛋白收縮分析 (Collagen contraction assay)中也可以發現，當正常頰黏膜纖維母細胞處理檳榔鹼後，產生收縮膠體能力，顯示其已分化成為肌纖維母細胞。接著透過染色質免疫沉澱技術 (Chromatin immunoprecipitation, ChIP)和報導基因分析 (Luciferase-based reporter assay)進一步證實，檳榔鹼誘導轉錄因子 ZEB-1的表現增加後，結合至α-SMA基因之啟動子(promoter)的E-Box序列上，促使α-SMA的表現。相反的若將正常頰黏膜纖維母細胞以RNA干擾技術抑制 ZEB-1 的表現後，則可以明顯看到隨著 ZEB-1表現降低，α-SMA的蛋白質表現也隨之減少，而在後續的膠體收縮分析和報導基因分析也能看到相同的結果。了解 ZEB-1在口腔黏膜下纖維化發生過程中的角色，提供了研發治療口腔黏膜下纖維化之藥物一種新的方向。

There are more than two million Taiwanese with betel nut consumption habit. This behavior causes several oral diseases including oral submucous fibrosis (OSF) and oral cancer but the pathogenesis of betel nut induced OSF is still not fully understood. Myofibroblasts, which identified as alpha-smooth muscle actin (α-SMA) positive cells, are frequently found in fibrotic tissues and participate in fibrogenic process through collagen secretion and induction of tissue contraction. Arecoline is the well-known alkaloid natural product in betel nut and has been suggested to induce OSF and oral cancer.
Here we investigated the role of arecoline in the differentiation of myofibroblasts from normal buccal mucosal fibroblasts (BMFs). We first determined the effect of arecoline in cell growth of firbolasts and the IC50 is 72.4±27.9 μg/ml. We further observed that arecoline could increase cell proliferation in BMFs at the concentration of 10-20μg/ml and induce the expression of α-SMA and vimentin, a marker of mesenchymal cells, simultaneously. In order to understand the molecular mechanisms of arecoline-induced α-SMA expression, we focused on the molecules involving in epithelial-mesenchymal transition (EMT). By western blot, we found that the transcriptional repressor in EMT process, Zinc finger E-box binding homeobox 1 (ZEB-1), were induced by arecoline simultaneously with α-SMA expression in normal BMFs. Through collagen contraction assay, we identified that arecoline induced myofibroblast activity in normal BMFs. Arecoline also could increase α-SMA expression in normal BMFs through ZEB-1 by directly binding to E-box domain of α-SMA promoter. By RNA interference, we further demonstrated that knockdown of ZEB-1 could decrease arecoline-induced α-SMA expression, as well as the collagen contraction in normal BMFs. Understanding the role of ZEB-1 in arecoline-induced OSF may provide a new insight in the new drug development for treating OSF disease.

緒論 1
台灣口腔癌現狀 1
口腔癌前病變成因(Oral precancerous lesions) 2
口腔黏膜下纖維化(Oral submucous fibrosis, OSF) 2
檳榔鹼(Arecoline) 3
肌纖維母細胞特性(Myofibroblast) 4
上皮-間質細胞轉換過程(Epithelial-mesenchymal transition, EMT) 5
EMT 轉錄因子 (EMT Transcription factor) 7
研究動機 9
實驗流程 10
材料與方法 12
A.材料 12
1.選用細胞 12
2.試劑 12
3.抗體 15
4.耗材&其他 17
5.RNAi 基因表現干擾技術病毒株 17
6.儀器設備 18
B.方法 19
1.藥物對於細胞增生以及耐受性測試 19
2.透過免疫螢光染色來觀察目標蛋白質的表現量 19
3.蛋白質濃度測定 21
4.藉由西方墨點法 (western blot)分析細胞內蛋白質表現 21
5.Total mRNA萃取 22
6.反轉錄作用 (Reverse transcription, RT-PCR) 23
7.定量即時聚合酶鏈鎖反應 (Real-time PCR, qPCR) 24
8.染色質免疫沉澱法 (Chromation immunoprecipitation, ChIP) 25
9.螢光素酶基因報導分析 (Luciferase-based Reporter Assay) 28
10.膠原蛋白膠體收縮分析 (Collagen contraction assay) 30
11.RNAi核糖核酸基因表現干擾技術 (shRNA knockdown) 31
結果 32
檳榔鹼 (Arecoline)對於正常頰黏膜纖維母細胞的影響 32
α-SMA、EMT相關轉錄因子及膠原蛋白在頰黏膜纖維母細胞表現情形 32
檳榔鹼誘導正常頰黏膜纖維細胞中α-SMA和EMT 轉錄因子ZEB1表現 33
頰黏膜纖維母細胞長期以低劑量檳榔鹼培養誘導α-SMA以及 ZEB1表現 34
長期低劑量檳榔鹼處理正常頰黏膜纖維細胞誘導產生肌纖維母細胞活性 35
短期內不同劑量檳榔鹼誘導頰黏膜纖維母細胞中 α-SMA及 ZEB1表現 36
短期內檳榔鹼處理誘導頰黏膜纖維母細胞具有肌纖維母細胞之活性 37
檳榔鹼在頰黏膜纖維母細胞中誘導ZEB1提高α -SMA promoter的活性 38
檳榔鹼誘導口腔頰黏膜纖維細胞活化 α- SMA promoter大量表現 α- SMA 39
抑制 ZEB1的表現可降低檳榔鹼所誘導之 α-SMA表現 40
抑制 ZEB1的表現可降低檳榔鹼所誘導之肌纖維母細胞活性 41
調控ZEB1表現對於激活 α-SMA promoter啟動子活性及 α- SMA表現情形 42
討論 44
文獻參考 52
圖 57

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