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系統識別號 U0026-0812200913593108
論文名稱(中文) 缺氧調控CD151表現對癌細胞附著能力的影響之探討
論文名稱(英文) Effect of hypoxia-regulated CD151 expression on cancer cell adhesion
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 95
學期 2
出版年 96
研究生(中文) 簡郡緯
研究生(英文) Chun-wei Chien
電子信箱 s3694409@mail.ncku.edu.tw
學號 s3694409
學位類別 碩士
語文別 中文
論文頁數 59頁
口試委員 召集委員-李正昌
口試委員-楊孔嘉
指導教授-蔡少正
中文關鍵字 CD151  缺氧  腫瘤 
英文關鍵字 CD151  tumor  hypoxia 
學科別分類
中文摘要 CD151是一種膜蛋白,屬於tetraspanin 成員中的一員。目前CD151被發現可以調控細胞的附著能力與運動能力,並且與腫瘤的惡性化有高度的相關性。然而,卻沒有任何研究針對腫瘤細胞與正常細胞的CD151表現量作探討。在腫瘤組織中,CD151被認為可能扮演著腫瘤轉移抑制者的角色,而缺氧則是可以促進腫瘤的轉移。因此我們假設在缺氧的情況下所引發的腫瘤轉移,可能是缺氧壓力藉由負調控CD151的表現量而抑制了CD151的功能。在我們的研究中使用配對的大腸直腸檢體,也發現了在腫瘤部位的CD151表現量低於配對的正常組織,同時也觀察到缺氧誘導因子-1在腫瘤部位的高度表現。利用了生物資訊方法分析之後,發現在CD151基因的驅動子區域有缺氧反應序列的存在。而利用大腸直腸癌細胞株處以缺氧或是化學性模擬缺氧處理之後,可以觀察到隨著缺氧誘導因子-1表現上升,CD151表現量明顯下降。而在驅動子活性分析的實驗中,也發現CD151基因的缺氧反應序列在缺氧環境下其轉錄活性會下降,確認了在缺氧環境中CD151基因會受到缺氧誘導因子-1的負調控。更進一步的實驗證實,缺氧處理之後CD151表現量下降同時也引起細胞的附著能力的降低,使得細胞對於胞外基質貼附變差。這樣的過程可能是促使腫瘤細胞在缺氧的壓力之下,易於離開胞外基質並且更容易轉移的準備步驟。總結以上的發現,我們證明了缺氧會透過缺氧誘導因子-1抑制CD151的表現,並且使得腫瘤細胞附著能力變差,進而可能增加了細胞從基底膜的分離而使腫瘤轉移更容易發生。
英文摘要 CD151 is a member of tetraspanin family that plays important roles in cell adhesion and motility. However, the regulation of CD151 expression in normal and cancerous cells has not yet revealed. In cancer tissue, it is suggested that CD151 acts as a metastasis-suppressor while hypoxia has been shown to be a factor of causing metastasis. We hypothesize that hypoxia-induced cancer cell metastasis may be mediated via deregulation of CD151. Indeed, colon cancer cells expressed less CD151 than its normal counterpart, which is inversely correlated with the expression of hypoxia-inducible factor-1 (HIF-1). By using bioinformatic approach, we identified a putative hypoxia response element (HRE) in the promoter region of human CD151 gene. Treatment of colon cancer cells with hypoxia or chemicals that are known to increase accumulation of HIF-1 resulted in decreased CD151 expression. Promoter activity assay further demonstrated that the predicted putative HRE was pivotal for HIF-1-dependent downregulation of CD151 expression. Furthermore, inhibition of CD151 expression by hypoxia resulted in decreased cancer cell adhesion to laminin-coated matrix, a process that enables cancer cell to detach from its surrounding matrix and be ready for metastasis. In conclusion, we demonstrated that hypoxia downregulates the expression of CD151 thus enhances the detachment of cancer cells from its basement membrane protein, an important first step for metastasis.
論文目次 目錄………………………………………………………………… 1
圖錄………………………………………………………………… 3
中文摘要…………………………………………………………… 4
Abstract…………………………………………………………… 5
緒論………………………………………………………………… 6
實驗材料與方法…………………………………………………… 15
材料: ……………………………………………………………… 15
大腸直腸癌病人腫瘤組織與正常組織的收集…………………… 15
實驗方法: ………………………………………………………… 15
組織蛋白質萃取…………………………………………………… 15
免疫組織染色……………………………………………………… 16
免疫組織染色定性分析…………………………………………… 17
細胞培養…………………………………………………………… 18
缺氧處理…………………………………………………………… 18
萃取細胞蛋白質…………………………………………………… 19
蛋白質濃度分析…………………………………………………… 19
西方轉漬法………………………………………………………… 20
細胞轉殖…………………………………………………………… 21
螢光酵素分析……………………………………………………… 22
缺氧對細胞附著影響試驗………………………………………… 23
細胞附著能力試驗………………………………………………… 23
細胞連結試驗……………………………………………………… 25
統計分析…………………………………………………………… 25
結果………………………………………… ………………………27
A. CD151在腫瘤組織的表現量低於正常組織 27
B. CD151的表現量與缺氧誘導因子-1的表現量為負相關 28
C. 缺氧處理大腸直腸癌細胞株SW480促使CD151表現量下降 28
D. 缺氧處理影響在轉錄層次來降低CD151的mRNA 29
E. 缺氧可以透過缺氧誘導因子-1調控CD151的轉錄 30
F. 缺氧壓力使細胞貼附數目降低 30
G. 缺氧所導致的CD151下降減少了細胞的附著能力 31
H. 缺氧所導致的CD151下降減少了細胞的連結能力 33
討論………………………………………… 34
參考文獻………………………………………… 40
附圖………………………………………… 45
附錄: 溶液與藥品的配置………………………………………… 56
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