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系統識別號 U0026-0812200911100074
論文名稱(中文) 三價砷對角質細胞特性的影響
論文名稱(英文) The Effect of Arsenite on the Properties of Keratinocyte Cell Cultures
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 92
學期 2
出版年 93
研究生(中文) 黃勁天
研究生(英文) Jin-Tian Huang
學號 s1691404
學位類別 碩士
語文別 中文
論文頁數 83頁
口試委員 口試委員-林淑華
口試委員-張文粲
指導教授-吳華林
口試委員-施桂月
中文關鍵字 角質細胞  三價砷  細胞爬行  細胞附著 
英文關鍵字 arsenite  cell migration  cell adhesion  keratinocyte 
學科別分類
中文摘要   砷是一種廣泛存在於環境中的元素,並且是一個很嚴重的環境汙染毒物。有很多的相關研究都證明砷與許多的癌症具有高度的關聯性,其中最主要的是與皮膚癌的相關性。但另一方面,在過去幾世紀以來,各種的砷化物都有應用在疾病上的治療。甚至在中國大陸,利用三價砷治療急性前骨髓性白血病的研究已有臨床上的療效。砷化物對於醫療上的應用始終具有兩面的評價,而且對於三價砷所促進的癌化現象以及癌症上的治療,其機制也尚未明瞭。在本篇論文的研究當中,我們利用低濃度的三價砷(0.05 ~ 1.0 �M)以長時間的方法去處理人類的角質細胞(HaCaT 細胞,一種自發性不朽的角質細胞株),我們可以發現三價砷處理的細胞會變得具有較高的吸附細胞外間質(extracellular matrix)之能力,特別是針對細胞外間質fibronectin。此外,我們在細胞形態上的觀察也發現三價砷處理的細胞變的較為平貼於底部。更進一步的,我們利用兩種實驗去試驗細胞的爬行能力,可以發現在刮傷試驗(scratch wound assay )中,於光學顯微鏡下的觀察,三價砷處理的細胞往沒有細胞的區域爬行的速度是比較快的。另外在Boyden chamber assay中,我們同樣可以觀察到三價砷處理過的細胞,爬過含有孔洞的膜之另一邊的數量是比較多的。因此可以知道三價砷處理的細胞,其爬行能力較對照組的細胞為高。我們也進一步的利用不同種的細胞內訊息傳遞路徑的抑制劑去探討三價砷處理的細胞是經由何種調控機制去影響細胞的爬行,結果發現可能是經由ERKs及p38這兩種訊息傳遞路徑去影響三價砷處理的角質細胞之爬行。此外,藉由西方點墨法以及反轉錄聚合酶連鎖反應,我們測知處理三價砷的角質細胞其cadherin及uPA的蛋白或基因表現量是較低的,而paxillin和MMP-2的蛋白或基因表現量是則是較高的。Cadherin和paxillin是與細胞和細胞或底部基質的吸附能力有相關而MMP-2和uPA是與腫瘤細胞的侵略能力(tumor invasion)有相關。由以上結果,我們可以推測三價砷處理的角質細胞可能較具移動能力也較具侵略性。


英文摘要   Exposure to arsenic, a ubiquitous and toxic environmental pollutant, is associated with an increased incidence of skin cancer. On the other hand, arsenic also serves as a drug for the treatment of acute promyelocytic leukemia. However, the mechanism(s) associated with arsenite-mediated carcinogenesis and cancer therapies at low concentrations of exposure remains elusive. In our studies, we found that long-term exposure of HaCaT cells, an spontaneously immortalized keratinocyte cell line, with low concentrations of arsenite(0.05-1.0μM) could enhance adhesion ability of HaCaT cells to fibronectin. We also observed the morphology of arsenite-treated HaCaT cells became more flattened. Furthermore, in scratch wound and Boyden chamber assay models, we observed that arsenite-treated HaCaT cells were more motile than control cells. To investigate the mechanisms that may involve in the increase of migration activity of arsenite-treated HaCaT cells, we used different inhibitors of signal transduction pathways to dissect the possible mechanisms involved in the migration of arsenite-treated HaCaT cells. The results showed that ERKs and p38 may be involved in the migration event of arsenite-treated HaCaT cells. By using western blot and RT-PCR, we found that cadherin and uPA were down-regulated and paxillin and MMP-2 were up-regulated in arsenite-treated HaCaT cells. Cadherin and paxillin play important roles in cell-cell adhesion and cell-substratum adhesion. MMP-2 and uPA are proteolytic enzymes which play major roles in tumor invasion. These results imply that arsenite-treated HaCaT cells may be more motile and aggressive.


論文目次 中文----------------------------------------------1
英文摘要------------------------------------------3
誌謝----------------------------------------------4
目錄----------------------------------------------5
圖、附表目錄--------------------------------------8
縮寫檢索表----------------------------------------9
藥品及材料---------------------------------------11
儀器---------------------------------------------18
緒論---------------------------------------------20
研究動機-----------------------------------------24
材料與方法---------------------------------------25
一、HaCaT細胞的培養------------------------------25
A. HaCaT細胞之繼代培養---------------------------26
B. 冷凍保存細胞----------------------------------27
C. 解凍細胞的方法--------------------------------27
D. 細胞計數--------------------------------------28
二、西方點墨法 ( Western blot )------------------28
A. 收取細胞溶解液--------------------------------28
B. 蛋白質濃度測定--------------------------------29
C. 西方點墨法------------------------------------30
三、反轉錄-聚合酶連鎖反應 ( reverse transcription
–polymerase chain reaction; RT-PCR)-------------35
A.萃取細胞全部的RNA------------------------------35
B.DNase處理--------------------------------------37
C.反轉錄反應 ( reverse transcription; RT )-------38
D.聚合酶連鎖反應---------------------------------39
E.瓊脂膠電泳 ( agarose gel electrophoresis )-----40
四、角質細胞之特性分析---------------------------41
A.細胞形態的觀察---------------------------------41
B.細胞對細胞外間質( extracellular matrix; ECM )
黏著試驗( adhesion assay )-----------------------42
C.細胞爬行能力試驗 ( cell migration assay )------44
【a】刮傷試驗 ( scratch wound assay )------------44
【b】Boyden chamber assay------------------------45
D.細胞增殖試驗 ( cell proliferation assay )------47
五、細胞內訊息傳遞路徑之研究---------------------48
六、免疫螢光染色法 ( immunofluororescent stain )-49
七、Scratch wound assay之定量分析----------------50
結果---------------------------------------------52
一、長時間處理下,三價砷對角質細胞形態上的影響---52
二、長時間處理下,三價砷對角質細胞吸附細胞外間質的
影響---------------------------------------------52
三、長時間處理下,三價砷對角質細胞爬行能力的影響-53
四、長時間處理下,三價砷對角質細胞增殖的影響-----54
五、探討長時間處理下,三價砷促進細胞爬行能力之訊息
傳遞路徑-----------------------------------------54
六、長時間處理下,三價砷對角質細胞基因及蛋白質表現
上的影響-----------------------------------------55
討論---------------------------------------------57
圖-----------------------------------------------63
附表---------------------------------------------76
參考文獻-----------------------------------------78
自述---------------------------------------------83
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