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系統識別號 U0026-3008201613400700
論文名稱(中文) 口腔鱗狀細胞癌DNA甲基化調控異常研究
論文名稱(英文) DNA methylation deregulation in oral squamous cell carcinoma
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 104
學期 2
出版年 105
研究生(中文) 洪崇翊
研究生(英文) Chong-Yi Hong
學號 T46031046
學位類別 碩士
語文別 中文
論文頁數 62頁
口試委員 指導教授-黃則達
共同指導教授-王東堯
口試委員-謝達斌
口試委員-黃振勳
中文關鍵字 焦磷酸測定  AQP5  DNA甲基化 
英文關鍵字 Pyrosequencing  AQP5  DNA methylation 
學科別分類
中文摘要 口腔鱗狀細胞癌(口腔癌)居台灣癌症發生率第五名,且有逐年上升的趨勢;平均死亡年齡為54歲,較其他癌症早10年以上,其中嚼檳榔、吸菸、喝酒是造成口腔癌的主因。口腔癌病患就診時常為晚期之第三、四期,晚期病患也有較高之死亡率;因此早期篩檢在口腔癌中有其重要性。目前的篩檢方式視診、觸診、及確定診斷之切片檢查。口腔癌癌化的機轉逐漸被研究的同時,顯示其為多基因體變異,所累積起來的癌化過程,而有許多特殊基因體的變異是可考慮作為辨識病灶特徵的生物標記。DNA甲基化是屬於表觀基因體調控的一種,當基因促進子位置的DNA甲基化調控異常時,常會發生抑癌基因的不表現或是致癌基因的表現,進而與癌症的發生相關。因此,DNA甲基化調控異常可以作為早期檢測之生物標記。AQP5是一種水通道蛋白,與眼淚、唾液以及一些肺部的分泌有關;近來的研究發現,AQP5促進子的DNA甲基化調控異常與肺癌的發生有很大的相關聯性,而本研究則是要探討口腔癌與AQP5促進子的DNA甲基化調控異常之關係,使用焦磷酸定序的方式測定口腔癌細胞的DNA甲基化程度,發現DNA甲基化程度都很高,接著用口腔癌及癌前病變病人檢體作焦磷酸定序測定,同樣也發現了癌前病變AQP5的DNA甲基化程度高於正常的組織,但口腔癌之DNA甲基化低於癌前病變,這些結果可以推論AQP5促進子的高度甲基化造成了AQP5於癌前病變低表現量,而後於口腔癌因DNA甲基化低而表現量增加,進一步推論AQP5有可能是致癌基因。
英文摘要 Oral squamous cell carcinoma (oral cancer) was ranked the fifth of cancer incidence and increasing annually in Taiwan. The carcinogenesis of oral cancer is deeply investigated, revealed the cumulative carcinogenesis at the genetic and epigenetic levels, and also the feasible biomarker in oral cancer. Oncogene could be expressed or tumor suppression gene repressed as result of DNA methylation deregulated of gene promoter region. Pyrosequencing assay was used to evaluate the methylation level in oral cancer cell lines and patient tissue samples, and AQP5 was hypermethylation in oral cancer cell lines and precancer tissue samples. The expression of AQP5 was low by using qPCR. We concluded that the hypermethylation of AQP5 in precancer could induce low expression of AQP5, and hypomethylation of AQP5 in oral cancer might be the oncogene in oral cancer carcinogenesis.
論文目次 中文摘要 I
英文延伸摘要 III
致謝 VIII
緒論 1
口腔癌 1
表觀基因體調控機制與癌症之間的關係 1
DNA甲基化 2
Aquaporin 5與癌症之關係 4
研究動機 5
材料與方法 6
一、 癌細胞株培養(OECM-1、OC2、OC3、OML1、OML3、OCM3、OCMC、OCSL、Fadu、SAS、ME) 6
(1)繼代培養 6
(2)冷凍並保存細胞 7
(3)細胞解凍 8
(4)細胞計數 8
二、 DNA 甲基化調控分析之相關實驗 9
(1) 癌細胞株DNA 萃取 9
(2) 檢體 11
(3) 亞硫酸鹽轉換(Bisulfite Conversion) 11
(4) Primer 設計 12
(5)聚合酶連鎖反應(Polymerase Chain Reaction) 13
(6)焦磷酸定序(Pyrosequencing) 14
三、RNA表現量之分析 16
(1) RNA純化(RNA Purification) 16
(2) RNA定量 17
(3)反轉錄酶反應(Reverse Transcription) 17
(4)聚合酶連鎖反應(Polymerase Chain Reaction) 19
(5)洋菜膠體電泳分析(Agarose gel electrophoresis) 20
(6) 即時定量PCR(qRT-PCR) 21
實驗結果 23
討論 25
結論 29
參考文獻 30
附圖 32
補充資料 38
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21. Jung, H.J., et al., Aquaporin-5: a marker protein for proliferation and migration of human breast cancer cells. PloS one, 2011. 6(12): p. e28492.
22. Zhang, Z., et al., Expression of aquaporin 5 increases proliferation and metastasis potential of lung cancer. J Pathol, 2010. 221(2): p. 210-20.
23. Lin, T., et al., MicroRNA-143 as a tumor suppressor for bladder cancer. The Journal of urology, 2009. 181(3): p. 1372-1380.
24. Shimizu, T., et al., Methylation of a panel of microRNA genes is a novel biomarker for detection of bladder cancer. European urology, 2013. 63(6): p. 1091-1100.
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27. 衛生福利部國民健康署,103年國人死因結果分析
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