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系統識別號 U0026-0812200911425021
論文名稱(中文) 缺氧誘導因子-1alpha調控CD151之探討
論文名稱(英文) Regulation of CD151 (plate-endothelial tetraspanin antigen 3, PETA-3) by hypoxia inducible factor-1alpha
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 93
學期 2
出版年 94
研究生(中文) 蔡秀真
研究生(英文) Shiou-Jen Tsai
電子信箱 safehelmet@yahoo.com.tw
學號 s3692107
學位類別 碩士
語文別 中文
論文頁數 79頁
口試委員 召集委員-湯銘哲
口試委員-劉校生
指導教授-蔡少正
中文關鍵字 缺氧  大腸直腸癌  缺氧誘導因子-1alpha  CD151 
英文關鍵字 HIF-1alpha  hypoxia  PETA-3  CD151  colon cancer 
學科別分類
中文摘要   缺氧(hypoxia)的定義指細胞或組織處在低於正常氧氣分壓的狀態下。當細胞遭遇到缺氧時,會造成許多細胞功能的改變,而促使細胞能適應這樣的環境,其中這樣的機制大部分可透過缺氧誘導因子-1alpha來調控。當缺乏氧氣時,細胞內的缺氧誘導因子-1alpha會與缺氧誘導因子-1beta結合形成二聚體,並結合到受其調控基因的DNA缺氧反應序列上,進而調控下游基因的表現。在臨床的研究報導指出,缺氧會造成腫瘤組織對於癌症治療上具有抵抗性,並且在許多的癌症中均發現缺氧誘導因子-1alpha過量表現的情形。先前我們實驗室利用生物資訊的方式,分析文獻中已被報導證實受到缺氧誘導因子-1alpha調控基因的HRE及其周圍序列,進而建構一個較長序列HRE模型,將人類及小鼠所有基因的啟動子區域與此模型做比對,結果得到許多可能受到缺氧調控的基因,CD151為其中之一可能受到缺氧調控的基因,並且這樣的想法在目前的文獻中尚未被探討。CD151是一個穿越細胞膜的蛋白(屬於tetraspanin superfamily的成員之一),過去研究指出CD151在許多的細胞中均有表現(大部分於上皮細胞、內皮細胞及血小板等),而CD151在細胞中所扮演的角色被認為與細胞的附著和細胞移動的能力相關,並且被認為參與在許多癌症侵襲轉移的過程中。首先,我們由臨床的檢體中發現CD151於同一大腸直腸癌病人腫瘤組織中的表現量明顯低於正常組織,由於觀察到這樣的現象,因此在接下來的研究我想探討缺氧是否會影響CD151基因的表現?藉由給予大腸直腸癌細胞株化學性模擬缺氧處理後,CD151不論是在mRNA或是在蛋白質的表現量均有明顯的減少,並且這樣的處理也會造成CD151啟動子的活性下降。綜合以上結果,我們證實了缺氧會經由缺氧誘導因子-1alpha來調控CD151基因的表現,而這樣的現象使我們推測缺氧會造成腫瘤細胞CD151表現量減少,使得細胞附著在細胞外基質的能力變弱,因而增加腫瘤細胞的移動能力,進而促進大腸直腸癌的侵襲轉移的過程。
英文摘要  Hypoxia stress has been shown to involve in several biological processes such as angiogenesis and tumorigenesis. The most rapidly growing region of solid tumors undergoe low oxygen tension because of an imbalance in oxygen supply and consumption and this process called hypoxia. Hypoxia inducible factor-1alpha(HIF-1alpha) is a transcription factor that regulates expression of hypoxia response genes. Previously we had constructed a conserved hypoxia response element (HRE) model by bioinformatics methods. By using this model to screen human, mouse, and rat promoter sequences, thousands of the genes containing HREs have been identified. In this study, a novel candidate gene, CD151, is selected for further characterization. CD151 is a member of the tetraspanin superfamily and broadly expressed by a variety of cell types, notably epithelial cells, endothelial cells, muscle cells, Schwann cells, megakaryocytes, and platelets. This molecule is noted for its strong molecular associations with integrins. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated that HIF-1alpha is elevated in colon cancer cells. Given the bioinformatic prediction that the promoter region of CD151 contains functional HRE, it is likely that expression of CD151 in colon cancer cells might be regulated due to elevation of HIF-1alpha. Thus, we aim to determine effects of hypoxia on CD151 expression and its pathophysiological roles on cancer development and progression. We find that expression of CD151 in tumor tissues is lower than that in normal tissue in colon cancer patients. In vitro study demonstrated CD151 protein and mRNA levels are decreased after desferrioxamine mimic chemical hypoxic treatment in colon cancer cell line SW480 and SW620. Promter activity assay further demonstrated that hypoxia treatment inhibits reporter system carrying the HRE of human CD151 promoter. The hypoxia-induced decrease in CD151 expression can be reversed by transfection of dominant negative form HIF-1alpha. Results from this study should provide novel information in elucidating effect of hypoxia on one of the prognostic marker, CD151, in patients with colon cancer.
論文目次 中文摘要..................................................1
Abstract.................................................. 3
誌謝...................................................... 5

目錄...................................................... 6
圖錄...................................................... 9
緒論...................................................... 11
實驗材料與方法............................................ 21
(I)材料:
臨床大腸直腸癌人體檢體之取得........................... 21
使用之細胞株及細胞培養方法............................. 22
(II)實驗方法:
A. 缺氧處理細胞........................................ 23
B. 細胞total RNA 之純化................................ 23
C. 反轉錄聚合酶連鎖反應
(Reverse transcription polymerase chain reaction; RT-PCR)...............................................

24
D. 製備小量質體DNA (Minipreparation of plasmid DNA).... 25
E. 製備大量質體DNA (Midipreparation of plasmid DNA).... 26
F. 免疫組織染色 (Immunohistochemistry)................. 27
G. 蛋白質濃度分析 (Lowry assay)........................ 29
H. 蛋白質電泳與西方轉漬法 (Western blotting)........... 30
I. 細胞轉殖 (Transient transfection)................... 31
J. 螢光酵素檢測 (Luciferase assay)..................... 33
K. 統計分析 (Statistical analysis)..................... 34
結果 .................................................... 35
A. C151於大腸直腸癌病人腫瘤組織的表現量低於正常組織.... 35
B. CD151於大腸直腸癌病人腫瘤組織及與距離腫瘤不同方向
(靠近胃及遠離胃)的正常組織中表現量...................
35
C. CD151於大腸直腸癌病人腫瘤組織及與距離腫瘤不同距離的正常組織中表現量.......................................
36
D. 大腸直腸癌病人臨床分期與CD151蛋白表現量的關係...... 36
E. 利用免疫組織染色觀察大腸直腸癌組織中CD151蛋白的表現.....................................................
37
F. 缺氧誘導因子-1蛋白於大腸直腸癌病人正常組織及腫瘤組織的表現的情形.........................................
37
G. DFO處理大腸直腸癌細胞株SW480及SW620促使CD151 mRNA表現量下降.............................................
38
H. DFO處理大腸直腸癌細胞株SW480及SW620促使CD151 蛋白質表現量下降...........................................
39
I. DFO處理可透過缺氧誘導因子-1促使細胞中CD151蛋白質的表現量減少.............................................
39
J. 利用生物資訊方式分析CD151驅動子所具有的HRE序列..... 40
K. 化學模擬缺氧處理後促使缺氧誘導因子-1蛋白質在細胞核中聚積的情形增加.......................................
40
討論 .................................................... 59
參考文獻 ................................................ 67
附錄: ...................................................
(一)溶液的配製 ........................................ 72
A.細胞培養相關溶液............................. 72
B.蛋白質分析相關溶液........................... 74
C. 細胞萃取相關溶液............................ 74
D. 質體製備相關溶液............................ 75
(二)使用藥品廠牌一覽表 .............................. 77
圖錄
圖一‧ CD151蛋白質於大腸直腸癌病人正常大腸組織及腫瘤組織中的現........................................
42
圖二‧ CD151蛋白質於大腸直腸癌病人靠近肛門處或是接近胃的正常大腸組織與腫瘤組織中的表現量..............
43
圖三‧ CD151蛋白質於大腸直腸癌病人腫瘤組織與距離腫瘤不同遠近的正常大腸組織中的表現量..................
44
圖四‧ 大腸直腸癌病人臨床分期與CD151蛋白質於腫瘤組織或距離腫瘤不同遠近的正常大腸組織中表現量之相關性..............................................

45
圖五‧ 免疫組織染色觀察CD151於人類胎盤中的表現........ 46
圖六‧ 免疫組織染色觀察CD151蛋白質於腫瘤組織及靠近胃或是接近肛門處的正常大腸組織與中的表現量..........
47
圖七‧ 免疫組織染色觀察CD151蛋白質於大腸直腸癌病人腫瘤組織與離腫瘤不同遠近的正常大腸組織中的表現量....
48
圖八‧ 缺氧誘導因子-1蛋白質於大腸直腸癌病人正常大腸組織及腫瘤組織中的表現............................
49
圖九‧ 大腸直腸癌細胞經DFO處理對於細胞核中缺氧誘導因子-1蛋白質表現的影響(聚積的現象)...............
50
圖十‧ 大腸直腸癌細胞株COLO 320DM經DFO處理後CD151 mRNA 的表現量........................................
51
圖十一‧ 大腸直腸癌細胞株SW480及SW620分別經DFO處理後CD151 mRNA 的表現量.............................
52
圖十二‧ 大腸直腸癌細胞株SW480及SW620分別經DMOG處理後CD151 mRNA 的表現量.............................
53
圖十三‧ 大腸直腸癌細胞株SW480經DFO處理後CD151 蛋白質的表現情形........................................
54

圖十四‧ 大腸直腸癌細胞株SW620經DFO處理後CD151 蛋白質的表現情形........................................
55
圖十五‧ 大腸直腸癌細胞株SW480經轉殖dominate negative缺氧誘導因子-1質體後給予DFO處理CD151蛋白質的表現..............................................

56
圖十六‧ CD151驅動子結構及所具有可能缺氧誘導因子-1結合的HRE序列.........................................
57
圖十七‧ 利用25個鹼基的CD151 HRE序列來分析DFO對於CD151啟動子活性的影響................................
58
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