臺灣博碩士論文加值系統

口腔黏膜下纖維化(oral submucous fibrosis, OSF)屬於一種口腔癌癌前病變。在台灣，這些病變的發生原因絕大部分是和嚼食檳榔、抽煙有關。治療OSF這類的口腔疾病多以外科切除病灶合併植皮手術，並進行術後張口復健，缺乏專一性的非手術性藥物治療。
肌纖維母細胞是一種可誘導組織間質內收縮，並且表現平滑肌肌動蛋白(-smooth muscle actin, -SMA)的細胞群，在口腔黏膜下纖維化時，肌纖維母細胞會參與膠原蛋白的分泌，造成口腔黏膜的纖維化。白藜蘆醇(Resveratrol)是一種在紅酒中被發現可抗氧化的多酚類化合物，動物研究中發現白藜蘆醇能藉由降低心肌纖維化(cardiac fibrosis)而達到保護心臟的作用。
本研究目的為探討白藜蘆醇是否可用於改善口腔黏膜纖維母細胞之纖維化程度，並探討其作用之分子機制。首先我們確定白藜蘆醇對於纖維化頰黏膜纖維母細胞(fibrotic buccal mucosal fibroblasts, fBMFs)的生長影響，抑制50%細胞生長的濃度(IC50)分別為(130  6.16) M和(139.1  19.9) M。而在膠原蛋白膠體收縮分析(Collagen contraction assay)中，可以觀察到當白藜蘆醇濃度為100uM時，膠體收縮有顯著地被抑制。
為了解由白藜蘆醇抑制膠體收縮的分子機制為何，我們把目標專注在上皮-間質細胞轉換過程(epithelial-mesenchymal transition, EMT)的誘導分子上。透過西方墨點法(Western blot)，我們觀察到白藜蘆醇可誘導口腔纖維母細胞中的EMT轉錄因子Zinc finger E-box binding homeobox 1(ZEB1)之蛋白質表現量下降，同時-SMA和S100A4的表現也隨著下降。藉由定量聚合酶鏈鎖反應，我們也發現白藜蘆醇可以抑制ZEB1之mRNA轉錄。接著我們利用亞硫酸鹽定序(bisulfite pyrosequencing) 發現到白藜蘆醇僅可些微增加ZEB1 啟動子區內CpG islands的DNA甲基化，推測增加DNA甲基化並非白藜蘆醇藥效的主要機制；利用染色質免疫沉澱技術(Chromatin Immunoprecipitation, ChIP)則發現白藜蘆醇可增加組蛋白3離胺酸27三甲基化蛋白(trimethylated histone 3 at lysine 27, H3K27me3)結合到ZEB1 啟動子區，並且白藜蘆醇可以增加纖維化頰黏膜纖維母細胞內之組蛋白甲基轉移酶EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit)及H3K27me3的表現。若以RNA干擾技術抑制EZH2的表現後，可以看到隨著EZH2表現降低，ZEB1的mRNA表現隨之增加。透過這些結果，我們發現白藜蘆醇可透過表觀遺傳學(epigenetic)的方式，調控ZEB1的表達，進而達到抑制口腔黏膜下纖維化的效果。

In Taiwan, the behaviors of betel nut consumption and smoking cause several oral diseases including oral submucous fibrosis (OSF), which is considered as an oral precancerous condition. Current management of OSF includes stopping the areca quid chewing habit, medication and surgical intervention. However, there is no specific non-surgical drug in treatment of OSF being developed. Myofibroblasts, which are cells with positive expression of a-smooth muscle actin (a-SMA), are participated in fibrogenic process through collagen secretion. Resveratrol is an antioxidant polyphenol found in red wine and has been suggested to have heart protection effect by reducing cardiac fibrosis in the animal study. Here we would like to evaluate the therapeutic effect of resveratrol in treatment of OSF and determine the underlying molecular mechanisms. We first determined the effect of resveratrol in cell growth of two fibrotic buccal mucosal fibroblasts (fBMFs) and the IC50 is (131.3 ± 6.2) mM and (139.1 ± 19.9) mM, respectively. We further demonstrated that resveratrol could decrease the contraction capability of these two fBMFs with collagen contraction assay at the concentration of 100mM. By western blot, we found that Zinc finger E-box binding homeobox 1(ZEB1), a transcriptional factor in epithelial-mesenchymal transition process, was repressed by resveratrol simultaneously with inhibition of a-SMA and S100A4 expression in fBMFs. With quantitative polymerase chain reaction method, we also found that resveratrol inhibited ZEB1 expression at transcription level. With bisulfite pyrosequencing, we found that resveratrol treatment only slightly increased DNA methylation on two CpG islands within ZEB1 promoter region in fBMFs. We also discovered that treatment of resveratrol significantly increased miR-200c expression, the well-known microRNA with ZEB1-targeting activity. We further found that resveratrol treatment in fBMFs increased the expression of enhancer of zeste 2 polycomb repressive complex 2 subunit(EZH2) and trimethylated histone 3 at lysine 27 (H3K27me3), the substrate of EZH2. With chromatin immunoprecipitation analysis, the binding of H3K27me3 on ZEB1 promoter in fBMFs was also enhanced by resveratrol. Finally, knockdown of EZH2 in fBMFs increased the expression of ZEB1. In conclusion, resveratrol displays an anti-OSF activity through epigenetic control of ZEB1 expression.

謝誌 I
中文摘要 III
Abstract V
緒論 p.1
1. 口腔癌 p.1
2. 口腔黏膜下纖維化 (Oral submucous fibrosis, OSF) p.2
3. 肌纖維母細胞 (Myofibroblast) p.2
4. 上皮-間質細胞轉換 (epithelial-mesenchymal transition, EMT) p.3
5. Zinc finger E-box-binding homeobox 1 (ZEB1) p.5
6. Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2)與trimethylated histone 3 at lysine 27 (H3K27me3) p.5
7. 白藜蘆醇 (Resveratrol) p.6
研究動機 p.8
材料與設備 p.9
1. 頰黏膜纖維母細胞 p.9
2. 試劑 p.9
3. 儀器設備 p.17
實驗方法 p.19
1. 細胞培養 p.19
2. 細胞增生以及藥物耐受性測試 p.19
3. 蛋白質濃度測定 p.20
4. 利用西方墨點法 (western blot) 偵測細胞內蛋白質表現 p.20
5. Total RNA萃取 p.21
6. 反轉錄反應(reverse transcription) p.22
7. 利用定量即時聚合酶鏈鎖反應(Quantitative real time polymerase chain reaction, qPCR) 偵測基因表現 p.23
8. 測定細胞內miR-200c表現 p.25
9. 膠原蛋白膠體收縮分析(collagen contraction assay) p.27
10. 染色質免疫沉澱 (Chromatin Immunoprecipitation, ChIP) p.28
11. 亞硫酸鹽反應(Bisulfite reaction) p.32
12. RNAi( RNA interference)核糖核酸基因干擾技術 p.33
實驗結果 p.35
1. 白藜蘆醇 (Resveratrol)對於纖維化頰黏膜纖維母細胞的影響 p.35
2. 白藜蘆醇處理纖維化頰黏膜纖維母細胞可抑制肌纖維母細胞的活性 p.35
3. 白藜蘆醇抑制纖維化頰黏膜纖維母細胞中ZEB1和纖維化相關因子的mRNA表現 p.36
4. 白藜蘆醇抑制ZEB1和纖維化相關因子的蛋白質表現具濃度及時間依賴性 p.37
5. 白藜蘆醇對於纖維化頰黏膜纖維母細胞中ZEB1 啟動子區域DNA甲基化的影響 p.38
6. 白藜蘆醇對於纖維化頰黏膜纖維母細胞中MicroRNA-200c(miR-200c)的影響 p.39
7. 白藜蘆醇透過增加H3K27me3的表現抑制ZEB1 啟動子的活性 p.40
8. 白藜蘆醇可誘導EZH2的表現 p.41
9. 抑制EZH2的表現可增加纖維化纖維母細胞內ZEB1 的表現 p.42
討論 p.44
參考文獻 p.54
圖表 p.62

圖目錄

圖一 白藜蘆醇(Resveratrol)對於纖維化頰黏膜口腔纖維母細胞(fibrotic Buccal Mucosa Fibroblasts, fBMFs) 之細胞毒性測試 p.62
圖二 白藜蘆醇對於纖維化頰黏膜纖維母細胞之肌纖維母細胞活性的影響 p.63
圖三 白藜蘆醇對於纖維化頰黏膜纖維母細胞內α-SMA及纖維化相關因子mRNA表現的影響 p.64
圖四 不同濃度之白藜蘆醇對於纖維化頰黏膜纖維母細胞內α-SMA及纖維化相關因子蛋白表現的影響 p.65
圖五 連續處理白藜蘆醇對於纖維化頰黏膜纖維母細胞內α-SMA及纖維化相關因子蛋白表現的影響 p.66
圖六 白藜蘆醇對於纖維化頰黏膜纖維母細胞內ZEB1啟動子區之DNA甲基化的影響 p.67
圖七 白藜蘆醇對於纖維化頰黏膜纖維母細胞內microRNA-200c表現的影響 p.68
圖八 白藜蘆醇增加纖維化頰黏膜纖維母細胞內H3K27me3結合到ZEB1啟動子區 p.69
圖九 白藜蘆醇對纖維化頰黏膜纖維母細胞內EZH2 mRNA表現的影響 p.70
圖十 白藜蘆醇對纖維化頰黏膜纖維母細胞內EZH2以及H3K27me3蛋白表現的影響 p.71
圖十一 抑制EZH2可增加纖維化頰黏膜纖維母細胞內ZEB1的表現 p.72
附錄一 p.73

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