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系統識別號 U0026-0812200911143639
論文名稱(中文) 研究Amphiregulin在口腔癌的致癌機轉
論文名稱(英文) Functional studies of Amphiregulin in oral carcinogenesis
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 92
學期 2
出版年 93
研究生(中文) 楊凱英
研究生(英文) Kai-Ying Yang
學號 t4691105
學位類別 碩士
語文別 中文
論文頁數 68頁
口試委員 指導教授-吳梨華
指導教授-蔡森田
召集委員-靳應臺
中文關鍵字 口腔癌 
英文關鍵字 Amphiregulin  oral cancer 
學科別分類
中文摘要   在美國每年約有50,000的口腔癌新病例被診斷出,而全世界每年約有500,000的口腔癌新病例。在近幾年內,在台灣口腔癌的罹患率已高居國人男性第四位。咀嚼檳榔可能是最主要的因素。雖然,目前口腔癌的危險因子已經清楚知道,但是致癌機轉的分子機制卻還不清楚。所以,我們利用微矩列基因分析比較正常口腔角質上皮與另外兩株口腔癌細胞株,其基因上的差異,從中我們發現Amphiregulin (AR) 在兩株口腔癌細胞株中的表現有異常。另外我們在一些文獻中發現AR是上皮生長因子(EGF)的一員,會刺激上皮細胞和纖維母細胞的生長。在乳癌、直腸癌都發現AR的表現都異常。近期的研究也發現,AR參與正調控明膠酶家族、促進腫瘤細胞侵襲的能力。不過,AR在口腔癌致癌機轉上的角色還不清楚。因此,我們建立一株可以穩定表現AR的口腔癌細胞;進而去探討當AR過量表現時對口腔癌細胞生長速率、遷徙與侵襲能力有何影響。在我們初步結果發現,AR的過度表現會加速口腔癌細胞的生長速度、侵襲能力與移動能力。雖然,在乳房癌細胞上發現,AR會正調控MMP9的表現進而增加侵襲能力;但是在口腔癌細胞上並無此現象。反倒是發現在表現AR的口腔細胞珠上MMP2表現量增加。另外,利用組織免疫染色法發現在部分口腔癌病人,腫瘤侵襲部位AR蛋白質的表現量較高。在臨床,利用RT-PCR發現AR的mRNA在在口腔癌病人早期就已經呈現一個過度表現的狀態。總結以上結果,AR在口腔癌細胞有調控促進生長、侵襲以及移動的角色。

英文摘要   Cancer of the oral cavity is newly diagnosed in approximately 50,000 Americans and 500,000 others worldwide each year. For several years, oral cancer has been the fourth cause of all cancers in Taiwanese men. Areca quid chewing was considered to be the major culprit. Although the risk factors for squamous cell carcinomas of the oral cancer are well recognized, little is known about the molecular mechanisms responsible for this malignancy. We used cDNA microarray analysis of normal oral keratinocytes and two oral cancer cell lines and found deregulation amphiregulin (AR) in oral cancer cell lines. According to the literature search, AR, a EGF (epidermal growth factor)-like ligand, stimulated the growth of epithelial cells and fibroblasts, and deregulation of AR was observed in breast cancer and colon cancer. Recent studies indicate that AR may be involved in upregulation of matrix metalloproteases, invasion of tumor cells, as well as hyperproliferation and inflammation observed in psoriasis. However, the role of AR in oral carcinogenesis remains elusive. We therefore established AR-overexpressing stable clones and analyzed the relationship of AR overexpression and the ability of oral cancer cells to undergo proliferation, migration, invasion, and to form colonies in soft agar. Overexpression of AR gene indeed increased the growth and invasion ability of oral cancers. Although AR may be involved in the upregulation of MMP-9 and the invasion ability in breast cancer cells, there was no correlation of MMP-9 increase with the presence of AR in KB oral cancer cells. Instead, the expression of MMP-2 was increased in the AR-expressing clones. Imunohistochemical staining of oral cancer specimens with anti-AR antibodies indicated that the AR protein was overexpressed in the invasive front of certain oral cancer tissues. Moreover, using semiquantitative RT-PCR, we found that the level of AR mRNA was increased in 50% of oral cancer tissues compared to their counterparts, and the percentage of such patients harboring the increased expression of AR mRNA increased with progression of the clinical staging. Taken together, AR may play a role in the growth, migration and invasion of oral cancer cells.

論文目次 項目 頁數
中文摘要 IV
英文摘要 VI
誌謝 VIII
圖目錄 X
表目錄 XI
英文縮寫對照表 XII
壹、緒論
一、什麼是口腔癌 2
二、口腔癌的發生率 3
三、口腔癌的致病因素 4
四、EGF 家族成員介紹 5
五、AR 的研究近況 7
六、研究動機、目的與策略 8

貳、材料與方法
一、正常口腔上皮細胞(NOK)之分離 11
二、口腔癌檢體來源及資料 11
三、北方墨點法 (Northern blot) 12
四、重組質體(pcDNA3.1-AR-SV40)之建構 14
五、細胞之培養 (Cell culture) 15
六、瞬間轉染 (Transient transfection) 15
七、細胞增生實驗 (Cell proliferation assay) 16
八、建立穩定轉染細胞株(Establishment of stable transfected cell line) 17
九、西方墨點法 (Western blot) 18
十、細胞及檢體total RNA的抽取 (Total RNA extraction from cell and tissue) 19
十一、反轉錄聚合酶連鎖反應 (Reverse transcription-polymerase chain reaction,RT-PCR) 20
十二、細胞生長速率 (Cell growth rate) 23
十三、侵襲能力測試 (Invasion ability assay) 23
十四、Zymography 24
十五、軟膠測試 (Soft agar assay) 25
十六、傷口癒合實驗 (Wound healing assay) 26
十七、免疫組織染色 (Immunohistochemistry) 27
參、結果
一、口腔癌病人AR的表現量 30
二、轉染AR基因後的細胞中AR mRNA蛋白質的表現 30
三、穩定表現AR細胞株之生長速率 (Growth rate) 31
四、穩定表現AR細胞株之移動速率 (Migration rate) 32
五、穩定表現AR細胞株之侵襲能力 (Invasion ability)32
六、穩定表現AR細胞株之軟膠測試 (Soft agar assay) 33
七、口腔癌病人之免疫組織染色 (Immunohistochemistry)
34
八、AR表現量與口腔癌進程的關係 35
肆、討論 36
伍、未來展望 40
陸、參考文獻 42
柒、圖 46
捌、表 59
玖、附錄 61
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