陳信銘臺灣大學：口腔生物科學研究所蔡旻汎Tsai, Ming-FanMing-FanTsai2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51262中文摘要 口腔黏膜下纖維化症（oral submucous fibrosis :OSF）是因嚼食檳榔而使口腔黏膜下結締組織產生過度纖維化的癌前狀態。在於許多器官如：肝、肺、皮膚…等的過度纖維化疾病，經研究發現都有過度的HDAC-2之表現。因此，將探討HDAC-2與OSF纖維化致病機轉的關聯性。首先本研究我們先分別收集了59例OSF病患與15例正常人的口腔頰黏膜組織標本，利用HDAC-2抗體進行免疫組織化學染色方法，偵測HDAC-2在這些標本之染色表現，結果發現15例正常組織其HDAC-2免疫染色呈現無或微弱之表現（發生率ICR=13.3%），而59例OSF組織則呈陽性反應（發生率ICR=96.6%），顯示OSF組織比正常者的HDAC-2有過度表現，且具統計學上差異的顯著（P=0.0045）。其次本研究使用OSF以及正常者口腔頰黏膜組織所培養出的纖維母細胞，抽取其蛋白質，以西方點墨法了解HDAC-2的表現，並利用抑制HDAC-2表現的藥物SAHA (suberoylanilide hydroxamic acid)，在不同的時間點，分別作用在OSF以及正常者的口腔纖維母細胞，結果發現OSF fibroblasts HDAC-2的表現比正常者有過度的表現（P<0.05）。在加入SAHA作用後，雖然OSF與正常者纖維母細胞的HDAC-2表現皆被抑制，然而在SAHA作用12小時後，正常者的纖維母細胞HDAC-2被抑制的程度比OSF者高，且有顯著的差異（P<0.05）。換言之，OSF纖維母細胞比正常者有更高HDAC-2的表現且不易被HDAC-2的抑制劑所抑制。因此，本研究結果發現，膠原蛋白的過度堆積與HDAC-2的過度表現及不易被其抑制劑抑制有關。ABSTRACT Oral submucous fibrosis (OSF) is a chronic debilitating disease and a pre-malignant condition of the oral cavity. It is characterized by a generalized submucous fibrosis. Epidemiological evidences strongly indicate the association of the areca quid (AQ) habit and OSF. It is regarded as a pre-cancerous condition. HDAC-2 (Histone deacetylase H2) has been evidenced to have over expression in some organs, such as lung, liver, and skin, but the underlying mechanisms remain to be clarified. In our study, we will discuss the pathogenesis between the severity of Oral submucous fibrosis (OSF) and the expression of HDAC-2 (Histone deacetylase H2). In the beginning, we collect 59 OSF specimens and 15 normal buccal mucosa that were examined the expression of HDAC-2 by immunohistochemistry (IHC). All patients received surgical excision of their precancerous at the Department of Oral and Maxillofacial Surgery, National Taiwan University Hospital, Taipei, Taiwan. The results of this study showed the HDAC-2 positive rate of OSF is significantly higher than that of normal oral mucosa (NOM) (P=0.0045).The incident rate of the OSF(ICR=96.6%）specimens, HDAC-2 is strongly expressed in the fibroblasts of connective tissue more than normal buccalmucosa（ICR=13.3%）specimens. In addition, the expression of HDAC-2 protein between OSF and NOM was quantified with the use of Western blotting and the results showed the same with IHC assay. Besides, SAHA (suberoylanilide hydroxamic acid) an HDAC-2 inhibitor was added to find the effect on OSF and NOM in a time- dependent manner by western blotting assay. And our studied indicated that after we added the SAHA for 12 hours this inhibitor is markedly reduced the expression of HDAC-2, especially in OSF (p<0.05). In other word, we can see the result suggests that In the OSF , HDAC-2 is strongly expressed in the fibroblasts of connective tissue protein more than normal buccal mucosa. And the result finds that HDAC-2 is higher expression and harder to be repressed in the OSF than in the NOM. Hence, the result suggests that over deposition of collagen maybe related to the over expression and hard to be repressed of HDAC-2 in the OSF.謝誌……………………………………………………………………Ⅰ 中文摘要………………………………………………………………Ⅱ Abstract…………………………………………………………Ⅲ 第一章 研究動機及目的……………………………………………1 第二章 口腔黏膜下纖維化症致病機轉的研究……………………4 一、前言 …………………………………………………………4 二、文獻回顧……………………………………………………5 第三章、實驗材料及方法……………………………………………10 3-1. 實驗組與控制組檢體的收集………………………………10 3-2.臨床資料的收集……………………………………………10 3-3.免疫組織化學染色（immunohistochemical stain）方法標本的固定和包埋………………………………………………10 3-4.陽性染色率判斷標準………………………………………14 3-5.統計方法……………………………………………………14 3-6.類頰黏膜纖維母細胞之培養………………………………14 3-7.繼代培養Subculture --（以10 cm培養皿為例）…………15 3-8.Counting and seeding………………………………………15 3-9. Cryopreservation --- Freezing cells………………15 3-10. Treatments ………………………………………………16 3-11.細胞毒性試驗………………………………………………16 3-12.西方點墨法 (Western Blotting Assay)…………………16 3-13統計分析 (statistical analysis)……………………19 第四章、結果…………………………………………………………20 4-1 免疫組織染色IHC結果……………………………………20 4-2 OSF之病歷資料與HDAC-2相關結果統計分析…………20 4-3 MTT測量結果………………………………………………21 4-4 SAHA藥物對抑制人類纖維母細胞與HDAC-2之相關 影響…………………………………………………………22 4-5 Collagen type I 與FLI-1在加入抑制HDAC-2藥物SAHA後之表現…………………………………………………………23 第五章、討論………………………………………………………24 第六章、總結………………………………………………………28 第七章、附表與附圖…………………………………………………30 參考文獻………………………………………………………………541780566 bytesapplication/pdfen-US口腔黏膜下纖維化症第一型膠原蛋白組蛋白去乙醯化oral submucous fibrosisHDAC-2collagen typeI[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51262/1/ntu-96-R94450018-1.pdf