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研究生:鍾雯如
研究生(外文):Wen-JuChung
論文名稱:鱗狀細胞癌抗原(SCCA)在腫瘤弱酸環境中的臨床意義
論文名稱(外文):Clinical implication of squamous cell carcinoma antigen(SCCA)overexpression in acidic tumor microenvironment
指導教授:吳昭良許耿福許耿福引用關係
指導教授(外文):Chao-Liang WuChao-Liang Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:口腔醫學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:59
中文關鍵詞:鱗狀上皮細胞癌抗原乳酸弱酸環境活性氧化物
外文關鍵詞:SCCAsLactic acidpH6.5ROS
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臨床上常用腫瘤標誌 ( Tumor marker ) 的濃度高低來預測病人的預後及追蹤病情。鱗狀上皮細胞癌抗原 ( Squamous Cell Carcinoma Antigen, SCCA ) 是目前較具臨床應用價值的腫瘤標誌之ㄧ。根據臨床的診斷,子宮頸癌患者之 SCCA 指數偏高者,通常較有不良的預後結果。在我們先前研究結果發現 SCCA 的高表達量,對於化學藥物會產生抗性,例如:藜蘆醇 ( Resveratrol ) 及含鉑化合物 ( Cisplatin )。但是SCCA在腫瘤細胞中所扮演的角色及分子機制仍不是很清楚。有研究指出,癌細胞生長速度遠大於正常細胞,其能量來源也偏好利用醣解作用取代正常細胞的有氧循環,此一現象被稱為〝瓦氏效應〞。醣解作用的產物會導致乳酸的堆積,進而造成腫瘤細胞外環境的酸化。也有文獻指出,腫瘤細胞外環境的酸化會促進腫瘤的生成和轉移。在我們過去的研究發現, SCCA會去抑制溶解體蛋白酶酵素 L 的活性 ( Cathepsin L ) 並且可以拮抗癌細胞的死亡。在本次的研究目的發現,癌細胞處在弱酸的環境下,會促使SCCA表現量增加, 對於化學藥物如藜蘆醇 ( Resveratrol ) 及含鉑化合物 ( Cisplatin ) 也會產生抗性。然而酸性環境會引起活性氧化物 ( Reactive oxygen species, ROS ) 的產生,進而引起 MAPK 分子的訊息傳遞以及轉錄因子CREB的磷酸化。我們將癌細胞處理在弱酸的環境下,同時加入活性氧清除劑,可以抑制 SCCA 表現量也可以看到轉錄因子 CREB 的磷酸化會降低。我們利用軟體分析 SCCA1 啟動子結合位區域,發現有轉錄因子CREB 的結合位。我們預測酸可能透過活性氧化物的增加,促進轉錄因子 CREB 的磷酸化進而增加轉錄的能力。由以上結果可知,癌細胞處在弱酸的環境下對於化學藥物產生抗性,可能是透過活性氧化物 ( ROS ) 將轉錄因子CREB 的磷酸化進而促使SCCA表現量增加。
In clinical, tumor markers can be use to help cancer diagnosis , predict the patient’s response to particular therapies and follow-up disease. The squamous cell carcinoma antigen (SCCA) is a tumor marker and has a clinical value. In clinical diagnosis, patients with higher expression of SCCA show poor prognosis. However, SCCA in clinical use was more than 20 years, but the biological function and mechanism still remain unclear. Our previous studies, we found that overexpression of SCCA led to drug resistance, such as RSV (Resveratrol) and Cisplatin. Cancer cells usually grow faster than normal cells. In contrast to normal differentiated cells, which rely primarily on mitochondrial oxidative phosphorylation to generate the energy needed for cellular processes, most cancer cells instead rely on aerobic glycolysis, a phenomenon termed the “Warburg effect”. Many cancer cells consume glucose and produce lactic acid and cause acidification of the tumor extracellular environment. Acidification of the tumor extracellular environment may promote tumor formation and metastasis. In our previous studies showed the SCCA is a lysosomal proteases inhibitor and could inactivate cathepsin L and anti-cell death. This study we show the cancer cells cultured in low pH (Acidosis) media, increase SCCA expression. The cancer cells culture in low pH (Acidosis) media display drug resistance of RSV (Resveratrol) and Cisplatin. The acidosis enhances the formation of reactive oxygen species (ROS) and induces MAPK signaling pathway and transcription factor, CREB phosphorylation. The cancer cells treated with ROS scavenger reduce SCCA expression and CREB phosphorylation in acidosis condition. We found that, there is a CREB binding region in the promoter region of SCCA1 gene. These results suggested that acidosis increased phosphorylation of the transcription factor CREB via ROS, and them increased its transcriptional activity. Taken together, we suggest cancer resist drug-induced cell death in acidosis condition is through ROS-increased CREB transcriptional activity to promote SCCA1 gene expression .
考試合格證明........................................... I
中文摘要 .............................................. II
Abstract ............................................. IV
誌謝 .................................................. VI
總目錄 ................................................ VIII
圖目錄................................................. XI
縮寫 .................................................. XII
第一章 緒論 ............................................ 1
1. 鱗狀上皮細胞癌抗原
1.1 臨床意義 .......................................... 1
1.2 鱗狀上皮細胞癌抗原簡介.............................. 1
1.3 生物功能性 ........................................ 2
2. 腫瘤的微環境
2.1 腫瘤的形成......................................... 3
2.2 瓦氏效應 ( Warburg effect ) 與腫瘤的關係............ 3
2.3 腫瘤的酸性環境..................................... 4
3. 活性氧化物 ( Reactive oxygen species, ROS )
3.1 活性氧化物 ( ROS ) 簡介.............................. 5
3.2 活性氧化物 ( ROS ) 與生物體的代謝關係.................. 6
第二章 研究目的 ......................................... 8
第三章 材料與方法 ........................................ 10
一、 實驗材料
1.1 引子 ( Primer ) ................................. 10
1.2 抗體 ( Antibody ) ................................ 10
1.3 細胞株 ( Cell line ) ............................... 11
1.4 化學藥品及試劑組 ( Chemical reagent and assay kit )... 11
1.5 緩衝液 ( Buffer ) .................................. 14
二、 實驗方法
2.1 細胞株培養 ( Cell culture ) ......................... 19
2.2 細胞數目測定 ( Cell count ) ......................... 19
2.3 細胞冷凍儲存 ( Cell freezing ) ...................... 21
2.4 細胞實驗培養條件 ( Condition medium ) ................ 21
2.5 生物晶片分析 ( Microarray ) ......................... 22
2.6 西方墨點法 ( Western blot analysis ) ................ 22
2.7 互補DNA(Complementary DNA, cDNA )................. 23
2.8 聚合酶鏈鎖反應 ( Polymerase chain reaction, PCR ) ... 24
2.9 即時半定量聚合酶鏈鎖反應 ( Real time - PCR ) .......... 25
2.10 細胞存活測試 ( Cell Viability ) .................... 25
2.11 細胞免疫染色 ( Cell staining ) ..................... 26
2.12 免疫組織化學染色 ( Immunohistochemistry assay, IHC ) 27
2.13 流式細胞儀分析 ( Flow Cytometry analysis ) ......... 28
第四章 結果 ............................................. 29
一、HeLa cell 在弱酸環境下會誘導 SCCA1 與 SCCA2 基因的表現.. 29 二、HeLa cell 培養在弱酸環境下對於藥物會產生抗性............ 30
三、Lactic acid ( LA ) 和 L(+)-Lactic acid ( L ) 誘導 SCCA1
和SCCA2 蛋白質表現 .................................. 31
四、 HeLa cell 在弱酸環境下會引起活性氧化物 ( ROS ) 的形成...32
五、 弱酸環境下會增加 CREB 的磷酸化 ....................... 33
第五章 結論 ............................................. 34
第六章 討論 ............................................. 36
參考文獻 ................................................ 40
圖表 ................................................... 49

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