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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王淑美 | |
dc.contributor.author | Chia-Huei Chen | en |
dc.contributor.author | 陳佳徽 | zh_TW |
dc.date.accessioned | 2021-06-15T01:18:24Z | - |
dc.date.available | 2011-09-15 | |
dc.date.copyright | 2009-09-15 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-27 | |
dc.identifier.citation | Ahn, B.H., Kim, S.Y., Kim, E.H., Choi, K.S., Kwon, T.K., Lee, Y.H., Chang, J.S., Kim,
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42637 | - |
dc.description.abstract | 先前的研究指出:以酸性培養液 (pH = 6.6)處理肝癌細胞,會影響細胞黏附接
合 (adherens Junction; AJ)穩定度,造成E-cadherin/β-catenin complex 瓦解,使原本緊密接合的群聚細胞狀態轉變為扁平狀、移行能力增強的分散細胞。本篇研究則繼續探討酸性微環境,是否影響p120-catenin (p120ctn)酪胺酸 (tyrosine)或是絲胺酸 (serine)/蘇胺酸 (threonine)等不同的磷酸化修飾機制,進而影響cadherin/catenin junction complex 的瓦解。實驗結果顯示,與pH 7.4 控制組相比,pH 6.6 處理組於黏附接合處的p120ctn 螢光染色量明顯減少。利用流式細胞儀偵測細胞內鈣離子變化,發現pH 6.6 處理後細胞內鈣離子濃度有明顯上升,同時,我們也發現PKCα和PKCδ 的活化,其次,利用抑制劑抑制此二種PKC 激酶或使用RNAi 技術分別降低PKCα 及PKCδ 之蛋白質表現量,皆可阻止因為酸性處理造成接合處p120ctn染色的減少,證明PKC 活化確實與p120ctn 在黏附接合處消失有關。本實驗進一步利用免疫沈澱法觀察到pH 6.6 處理會造成p120ctn 絲胺酸磷酸化 (serine phosphorylation)程度下降,使用PKCd抑制劑rottlerin 及PKCa抑制劑Go6976 抑制不同PKC 異構物,能夠阻擋p120ctn 絲胺酸去磷酸化現象,這表示PKCδ 可能直接修飾p120ctn 絲胺酸殘基。同時,本研究使用去磷酸酶抑制劑calyculin A 預先處理,發現calyculin A 也可以阻止AJ 處p120ctn 螢光染色減少的現象,這暗示著絲胺酸/蘇胺酸去磷酸酶也可能參與pH 6.6 處理對於p120ctn 的修飾機制。此外,我們也觀察到pH 6.6 處理會增加p120ctn 酪胺酸磷酸化 (tyrosine phosphorylation),伴隨著E-cadherin/p120ctn complex 的結合力減弱,進一步利用Src 家族激酶抑制劑PP2 預先處理,結果顯示PP2 能夠避免p120 酪胺酸磷酸化程度上升,並使得p120ctn與E-cadherin 之結合增強。已知酸性培養液藉由活化Src 家族激酶並引起p120ctn酪胺酸磷酸化程度上升,我們也確認可能參與的成員,為c-Src,Fyn 及Yes,與p120ctn 的結合能力也會受到酸性pH 刺激而大幅增加。總結而言,本研究證實酸性培養液藉由活化Src 家族激酶以增加p120ctn 酪胺酸磷酸化和活化PKC 來降低p120ctn 絲胺酸磷酸化的機制,進而影響細胞黏附接合處p120ctn 穩定度。 | zh_TW |
dc.description.abstract | Previous studies have shown that acidic extracellular pH (pHe) increases the motility of hepatocellular carcinoma via disruption of the integrity of adherens junction, leading to the morphological changes from strong adhesion cluster to high motility, flattened and dispersed cells. The aim of this study was to investigate whether acidic pHe affected the cadherin/catenin complex via modifying the p120-catenin (p120ctn) at tyrosine or serine/threonine residues. In comparison to the continuous p120ctn immunostaining at cell-cell contact sites in pH 7.4-treated cells, weak and discontinuous p120ctn staining was observed in pH 6.6-treated cells. First of all, the increasing of intracellular calcium concentration under acidic pHe treatment was detected by flow cytometry. The second, immunoblotting showed that acidic pHe induced PKCα and PKCδ activation. The third, knocking down the PKCα and PKCδ by RNAi, respectively, could prevent acidic pHe-induced detachment of the p120ctn from cell junction, supporting the involvement of these PKC isoforms in regulation of junctional p120ctn.
Immunoprecipitation analysis further demonstrated that acidic pHe induced serine dephosphorylation of p120ctn. Furthermore, p120ctn serine dephosphorylation could be blocked by pretreatment with rottlerin, a PKCδ inhibitor. These observations suggested that the decreased staining of p120ctn at cell junction was induced by PKC activation. Also, acidic pHe-induced p120ctn detachment from cell-cell contact was prevented by a serine/threonine phosphatases inhibitor, calyculin A, implying that serine/threonine phosphatases might be involved in p120ctn modification. Further, we observed that acidic pHe increased the p120ctn tyrosine phosphorylation, which subsequently weakened the association of E-cadherin and p120ctn. Inhibition of Src family kinases by PP2 attenuated the acidic pHe-induced tyrosine phosphorylation of p120ctn and resumed the disassociation of E-cadherin/p120ctn. We further proved that acidic pHe activated Src family kinases, including c-Src, Fyn and Yes, which are known to increase p120ctn tyrosine phosphorylation. In conclusion, this study demonstrates that acidic pHe activates Src family kinases and PKCδ, resulting in tyrosine phosphorylation and serine dephosphorylation of p120ctn. Both modifications are contributed to the instability of p120ctn at AJ induced by acidic pHe. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:18:24Z (GMT). No. of bitstreams: 1 ntu-98-R96446011-1.pdf: 4232058 bytes, checksum: 12d9d7bf8dacb74aa9c790ba1d374327 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 誌謝.......................................................................................................................i
中文摘要...............................................................................................................ii Abstract..................................................................................................................iii 目錄.......................................................................................................................1 圖片索引...............................................................................................................3 第一章 緒論.......................................................................................................4 一、細胞外酸性pHe 對於腫瘤細胞之影響..................................................4 二、細胞的黏附接合與p120-catenin .............................................................5 三、蛋白質激酶C ...........................................................................................7 四、絲胺酸/蘇胺酸蛋白質去磷酸酶..............................................................8 五、Src 家族激酶調控黏附接合.....................................................................8 六、研究動機...................................................................................................9 第二章 材料與方法...........................................................................................11 一、細胞培養 (cell culture).............................................................................11 二、阻斷/活化訊息傳遞路徑以及實驗所需抗體..........................................11 三、西方墨點分析法 (western blotting).........................................................13 四、免疫沈澱法 (immunoprecipitation) .........................................................14 五、免疫螢光染色 (immunofluorescence microscopy) .................................15 六、電穿孔實驗 (electroporation method) .....................................................15 七、流式細胞儀分析 (flow cytometry) ..........................................................16 八、細胞移行實驗 (cell migration assay).......................................................16 九、統計分析 (statistical analysis) .................................................................17 第三章 實驗結果...............................................................................................18 一、酸性pHe 對於p120-catenin 分布之影響...............................................18 - 2 - 二、酸性pHe 引發細胞內鈣離子濃度上升、活化不同PKC 異構物........18 三、酸性pHe 藉由活化PKC 影響p120ctn 及E-cad 於黏附接合處分布 變化...........................................................................................................19 四、活化PKC 引發p120ctn 絲胺酸去磷酸化而降低E-cad/p120ctn 之 結合力.......................................................................................................20 五、酸性pHe 活化PKCδ,影響細胞移行能力............................................21 六、酸性pHe 可能活化絲胺酸/蘇胺酸蛋白質去磷酸酶影響p120ctn 分布變化...................................................................................................22 七、酸化處理活化Src 家族激酶...................................................................22 八、活化Src 家族激酶會降低E-cad/p120ctn 結合力..................................23 九、活化SFKs 增加p120ctn 酪胺酸磷酸化亦增加SFKs 與p120ctn 之結合 .................................................................................................................. 第四章 討論.......................................................................................................25 第五章 參考文獻...............................................................................................32 第六章 圖片及圖片說明...................................................................................41 附錄一 、120ctn 結構圖.....................................................................................56 附錄二 、模式圖.................................................................................................57 附錄三 、英文專有名詞縮寫.............................................................................58 | |
dc.language.iso | zh-TW | |
dc.title | 細胞外酸化處理對p120-catenin磷酸化之影響:PKC和Src的角色探討 | zh_TW |
dc.title | Effects of Extracellular Acidic pH on p120-catenin Phosphorylation: the Roles of PKC and Src | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王懷詩,吳建春,陳玉怜 | |
dc.subject.keyword | 細胞外酸性pHe,p120-catenin,PKCδ,Src,絲胺酸/蘇胺酸磷酸化,酪胺酸磷酸化, | zh_TW |
dc.subject.keyword | acidic pHe,p120-catenin,PKCδ,Src,serine/threonine phosphatases,serine phosphorylation,tyrosine phosphorylation, | en |
dc.relation.page | 58 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-27 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 解剖學暨生物細胞學研究所 | zh_TW |
顯示於系所單位: | 解剖學暨細胞生物學科所 |
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