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研究生:王源發
論文名稱:4-氨基聯苯誘導膀胱癌細胞移轉分子機制之探討
論文名稱(外文):Study of molecular mechanisms on 4-aminobiphenyl-induced motility of bladder cancer cells
指導教授:陳建成陳建成引用關係陳麗琴陳麗琴引用關係
學位類別:碩士
校院名稱:國立高雄師範大學
系所名稱:生物科技系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:73
中文關鍵詞:4-氨基聯苯膀胱癌侵襲基質金屬蛋白酶移行活性氧
相關次數:
  • 被引用被引用:0
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  • 下載下載:7
  • 收藏至我的研究室書目清單書目收藏:0
膀胱癌是泌尿系統最常見的惡性腫瘤,由於其易移轉的特性以及有限的治療,因此預後很差。4-氨基聯苯(4-aminobiphenl, 4-ABP)是一種聯苯基氨衍生物,常在香菸與偶氮染料發現的,為已知的人體致癌物。因此膀胱癌的發生常與職業暴露於4-氨基聯苯相關。基質金屬蛋白酶(Matrix metalloproteinases, MMPs)的高度表現,研究顯示與膀胱癌的增加,以及其分子具有密切相關性。然而,膀胱癌的致癌因子4-ABP,是否會誘導膀胱癌細胞的移行和侵襲能力增加,以及其分子機制還有待闡明。在本研究中,我們發現人類膀胱癌(TSGH-8301 cell)的移行和侵襲能力會隨著4-ABP處理濃度增加而增加。4-ABP誘導的膀胱癌細胞移行和侵襲能力同時會被活性氧清除劑乙酰半胱氨酸(Acetylcysteine, NAC)顯著的抑制,而隨著4-ABP處理濃度的增加會使磷酸化細胞外信號調節激酶(phosphorylated extracellular signal-regulated kinases, p-ERK)和MMP-2和9的表現會隨之增加。同時,我們使用NAC(ROS清除劑)及U0126(p-ERK抑制劑)處理後可以顯著的抑制MMPs的表現。以上結果顯示,人類致癌物4-ABP可以藉由ROS、p-ERK路徑,促進MMPs表現增加,進一步調控膀胱癌細胞的移轉能力。
Bladder cancer is the most common malignancy of the urinary system, due to its easy metastasis characteristics and limited therapeutic, resulting in poor prognosis. 4-aminobiphenyl (4-ABP) is a biphenyl amine derivative often found in smoking and azo dyes, as a known human carcinogen. Therefore, the incidence of bladder cancer is often associated with occupational exposure to 4-ABP related substance. Matrix metalloproteinase (MMPs) is highly expressed in bladder cancer, and studies have shown that an increase of bladder cancer is closely associated with the molecule. Whether the carcinogen 4-ABP could increase the migration and invasion abilities of bladder cancer cells, and its molecular mechanism remains to be elucidated. In our study, we found that in human bladder cancer (TSGH-8301 cell) cells, migration and invasion will be increased by 4-ABP in a concentration-dependent manner. 4-ABP-induced migration and invasion of bladder cancer will also be active oxygen scavenger acetylcysteine significant inhibition, but with 4-ABP treatment concentration will increase the phosphorylation of extracellular signal regulated kinase and the performance of MMP-2 and 9 will increase. We use the NAC (ROS scavenger) and U0126 (p-ERK inhibitor) can significantly inhibit expression of MMPs after treatment. Above results show that human carcinogen 4-ABP by ROS, p-ERK pathway, promote increased MMPs expression, to further control the motility of bladder cancer cell.
致謝 1
中文摘要 3
英文摘要 4
目錄 5
圖表目錄 7
壹、前言 8
1.1膀胱癌(Bladder cancer) 8
1.2 4-氨基聯苯(4-Aminobiphenyl, 4-ABP) 9
1.3基質金屬蛋白酶(Matrix metallopoateinases, MMPs) 10
1.4活性氧物質(reactive oxygen species, ROS) 11
1.5絲裂原活化蛋白激酶(Mitogen-activated protein kinases, MAPKs) 13
1.6研究目的(Specific aims of this study) 14
貳、材料與方法 16
2.1 實驗材料 16
2.2 實驗方法 19
2.2.1冷凍細胞之解凍 19
2.2.2細胞的冷凍保存 19
2.2.3細胞繼代培養 20
2.2.4細胞計數 20
2.2.5細胞存活率試驗(MTT assay) 20
2.2.6活性氧測定試驗( DCFH-DA assay) 21
2.2.7傷口癒合試驗(Wound-healing) 22
2.2.8細胞移行試驗(Cell migration) 22
2.2.9細胞侵襲試驗(Cell invasion) 23
2.2.10細胞溶解液(Cell Lysis) 24
2.2.11蛋白質濃度測定之(Bio-rad Protein assay) 25
2.2.12西方墨點法(Western blot) 26
2.2.13 RNA抽取 29
2.2.14反轉錄聚合酶連鎖反應(Reverse transcriotase-polymerase chain reaction,RT-PCR) 30
2.2.15細胞處理抑制劑之方式 32
參、結果 33
3.1 4-氨基聯苯對於TSGH-8301膀胱癌細胞株的細胞存活率影響 33
3.2 4-氨基聯苯誘導TSGH-8301膀胱癌細胞株移行能力的表現 33
3.3 4-氨基聯苯誘導TSGH-8301膀胱癌細胞株侵襲能力的表現 34
3.4 4-氨基聯苯誘導TSGH-8301膀胱癌細胞MMPs的表現 34
3.5 4-氨基聯苯可誘導TSGH-8301膀胱癌細胞株ROS的產生 35
3.6 4-氨基聯苯經由ROS誘導TSGH-8301膀胱癌細胞株的移行和侵襲能力增加 35
3.7 4-氨基聯苯誘導TSGH-8301膀胱癌細胞株MAPK路徑活化 36
3.8 p-ERK抑制劑降低4-氨基聯苯誘導TSGH-8301膀胱癌細胞株的移行和侵襲能力 36
3.9 ROS和p-ERK的上下游關係 37
3.10 p-ERK抑制劑會降低因4-氨基聯苯誘導TSGH-8301膀胱癌細胞株MMPs的蛋白質表現 37
肆、討論 38
4.1 4-氨基聯苯刺激下會誘導TSGH-8301膀胱癌細胞株MMPs表現 38
4.2 4-氨基聯苯經由ROS活化TSGH-8301膀胱癌細胞株內MAPK傳遞細胞訊息 39
4.3 4-氨基聯苯刺激下會誘導TSGH-8301膀胱癌細胞株產生ROS 39
4.4 4-氨基聯苯主要經由ROS-ERK路徑調控膀胱癌細胞移轉 40
伍、結論 41
陸、參考文獻 42
柒、附錄 70
7.1附錄一:4-ABP的特性http://www.trc-canada.com/index.php 70
7.2附錄二:MMPs的分類 71
7.3附錄三:The action principle of fluorescent dye DCFH-DA 72
7.4附錄四:Antibody dilution buffer and ration 73

圖表
Figure 1. Effect of 4-Aminobiphenyl on TSGH-8301 cell viability. 54
Figure 2. 4-Aminobiphenyl(4-ABP) affected the in vitro migration of TSGH-8301 cells. 56
Figure 3. 4-Aminobiphenyl(4-ABP) affected the in vitro invasion of TSGH-8301 cells. 57
Figure 4. Effect of 4-Aminobiphenyl-induced MMP-9 and MMP-2 expression in TSGH-8301 cell in a time dependent manner. 58
Figure 5. Effect of 4-Aminobiphenyl-induced MMP-1/2/9/10 mRNA expression in TSGH-8301 cell in a time dependent manner . 59
Figure 6. 4-Aminobiphenyl induces ROS production in a time dependent manner. 60
Figure 7. Suppression of ROS production by pharmacological inhibitors . 61
Figure 8. ROS is fundamental element during 4-ABP-induced cell migration in TSGH-8301 cell. 62
Figure 9. ROS is fundamental element during 4-ABP-induced cell invasion in TSGH-8301 cell. 63
Figure 10. Effect of 4-Aminobiphenyl-induced the phosphorylation levels of MAPK expression in TSGH-8301 cell in a time dependent manner. 64
Figure 11. p-ERK is fundamental element during 4-ABP-induced cell migration in TSGH-8301 cell. 65
Figure 12. p-ERK is fundamental element during 4-ABP-induced cell invasion in TSGH-8301 cell. 66
Figure 13. Involvement of MAPK pathways in 4-ABP-induced p-ERK expression. 67
Figure 14. Effect of p-ERK inhibitor U0126 on 4-ABP-induced MMP-2/9 expression. 68
Figure 15. Signaling transduction pathways involving in 4-aminobiphenyl regulating cell metastasis in TSGH-8301 bladder cancer cell line. 69

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