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研究生:蕭又誠
研究生(外文):Yu-cheng Hsiao
論文名稱:巨核細胞經過炭疽桿菌致死毒素處理之後,對細胞分裂相關蛋白在細胞中位置的影響
論文名稱(外文):The cellular localization of cytokinesis related proteins in Bacillus anthracis Lethal Toxin treated megakaryocytes
指導教授:孫德珊
指導教授(外文):Der-shan Sun
學位類別:碩士
校院名稱:慈濟大學
系所名稱:分子生物及細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
畢業學年度:94
語文別:中文
論文頁數:87
中文關鍵詞:細胞分裂相關蛋白炭疽桿菌致死毒素
外文關鍵詞:cytokinesis related proteinsBacillus anthracis Lethal Toxin
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炭疽桿菌致死毒素(LT)是炭疽桿菌一個重要的毒性因子,包括保護性抗原(PA)及致死因子(LF)。之前我們實驗室發現,不論是細胞的實驗或是在動物活體內,炭疽桿菌致死毒素均會阻斷巨核細胞的分化。而在巨核細胞形成血小板的過程中,需要透過分化的過程,包括細胞表面特殊抗原的表現(CD41b、CD61)及多倍體化(polyploidization)。多倍體化指的是在進行細胞週期時,會略過一些時期,例如:anaphase B、telophase及cytokinesis,並且DNA的含量會持續增加。最近研究指出,在巨核細胞進行多倍體化的過程期間,細胞中的Aurora kinases不僅會改變它們的位置,連含量都會有所改變。我們實驗室也證明了將human erythroleukemia cell line (HEL)前處理炭疽桿菌致死毒素後,再以TPA (12 - O - tetradecanoylphorbol - 13 - acetate)刺激,會造成actin-depolymerization factor - cofilin 及aurora B的表現量有所差異。因此,本實驗最重要的研究目的就是要解釋:炭疽桿菌致死毒素會阻斷巨核細胞的多倍體化, 是否藉由改變cofilin 與aurora B在細胞中的量與位置所造成的。我們使用共軛焦顯微鏡觀察,發現細胞中cofilin 的位置並無改變。卻發現當HEL在經由TPA刺激後,非活化的型態,phospho-cofilin會由細胞核轉移至細胞質中;若同時處理TPA與炭疽桿菌致死毒素後,又回復到跟處理DMSO 的控制組相同在核中表現。正常處理TPA 而分化的細胞是偵測不到aurora B 的,但當前處理炭疽桿菌致死毒素後,則發現aurora B c會恢復到原來的位置上,執行細胞分裂的功能。而細胞核中的p53 在處理DMSO 與TPA 這兩組的量很多,但前處理細胞毒殺劑量或低細胞毒殺劑量LT 這兩組,p53 的影像幾乎偵測不到。而由重組蛋白LT (W)與LT (M)的實驗可知,以上這些LT 造成的phospho-cofilin 位置的改變與p53 量的變化都與LF 的蛋白酵素活性有關,且低細胞毒殺劑量LT 也可以有相同的效果。証實LT 影響p53 在細胞核中的量減少及phospho-cofilin 在細胞質中與細胞核中位置的改變,是經由LF 切非MEKs 之另外的受質所造成的,至於這些現象與LT 抑制巨核細胞分化的關係,則有待進ㄧ步研究。
Anthrax lethal toxin (LT) composed of protective antigen (PA) and lethal factor (LF), is a critical virulence factor of Bacillus anthracis. Previously, we have found that LT could block megakaryocytopoiesis in vitro and in vivo. Megakaryocytes (MK) form platelets through differentiation including the expression of surface markers (CD41b and CD61) and polyploidization processes. Polyploidization means that the cell cycle skips some stages such as anaphase B, telophase, and cytokinesis, and keeps increasing DNA content. Previously, some study reported that Aurora kinases would change their cellular localization or expression level during polyploidization in megakaryocytes.Our previous studies demonstrated that the amount of cofilin (actin-depolymerization factor) was changed when treated with TPA (12-O-tetradecanoylphorbol-13-acetate) or TPA combined LT pretreatment in human erythroleukemia cell line (HEL). The major goal of this study is to examine whether LT blocks the polyploidization process of megakaryocytes by changing the subcellular localization and amount of cofilin and aurora B. We will detect the cellular distribution of cofilin and aurora B in TPA or TPA combined LT treated HEL cells by confocal microscope. The results showed that the localization of cofilin did not change, however, the localization of phospho-cofilin shifted from nucleus to cytoplasm when treated with TPA. In contrast, phospho-cofilin retained in nucleus as same as DMSO treated control cells when LT pretreatment. When pretreated with LT, the aurora B localized in its correct site to do the function of cytokinesis comparing with treated TPA only. We also found that the amount of p53 in cell nucleus was decreased when LT pretreatment. In conclusion, LT affectes the cellular localization of phospho-cofilin and the amount of p53 in cell nucleus and the catalytic activity of LF is required for all the effects. In recombinant protein experiment, the catalytic site of LT was necessary for the localization of phospho-cofilin and the amount of p53. The mechanism of how the changes of cellular distribution and the amount of cytokinesis related proteins influence megakaryocytic polyploidy is still unknown.
誌謝…………............................................................................................I
中文摘要..................................................................................................III
英文摘要...................................................................................................V
目錄.........................................................................................................VII
圖表目錄...............................................................................................VIII

緒論...........................................................................................................1
材料與方法...............................................................................................9
結果..........................................................................................................20
討論..........................................................................................................29
參考文獻..................................................................................................34
圖表..........................................................................................................40
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