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研究生:莊挺生
研究生(外文):Ting-Sheng Juang
論文名稱:研究炭疽桿菌致死毒素如何調控巨核細胞中cofilin的磷酸化
論文名稱(外文):Mechanisms of how Bacillus anthracis lethal toxin regulates phosphorylation of cofilin in 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:人類遺傳研究所
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:96
語文別:中文
論文頁數:75
中文關鍵詞:炭疽桿菌致死毒素
外文關鍵詞:cofilinBacillus anthracis
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炭疽致死毒素(Anthrax lethal toxin,簡稱為LT) 是炭疽桿菌(Bacillus anthracis)
的毒力因子,是由保護性抗原(protective antigen,簡稱為PA) 及致死因子(lethal
factor,簡稱為LF) 所組成的複合性毒素。PA能將LF帶到目標細胞的細胞質內,而
LF是個zinc-dependent metalloprotease,會藉由切割mitogen-activated protein kinase
(MAPK) kinase的N端而阻斷MAPK路徑。巨核細胞(megakaryocyte) 的成熟必須要透
過核內分裂(endomitosis) 促使多倍體細胞(polyploidy cells) 的生成。我們實驗室
先前運用二維電泳的方式證實了HEL (human erythroleukemia) 細胞處理TPA
(12-O-tetradecanoylphorbol-13-acetate) 產生多倍體的巨核細胞,會因為LT 的作用
而抑制HEL的分化,而這個過程中cofilin的表現量會改變。cofilin在細胞中是扮演著
調控actin的depolymerization (去聚合作用) 的功用,磷酸化的cofilin (p-cofilin) 會失
去調控actin 的能力。我利用西方墨點法(Western blotting) 去定量cofilin 與
phospho-cofilin,發現在HEL細胞處理TPA後p-cofilin表現量會上升,但先處理LT後再
給與TPA則p-colfin又會回復到原本的量。而且這cofilin的調控是會通過MAPK
pathway中的ERK與p38兩條訊息傳遞途徑來調控,其中ERK這條訊息傳遞途徑是通
過調控cofilin上游LIMK 1與phospho-LIMK (簡稱p-LIMK) 的表現量造成p-cofilin上
升。
Anthrax lethal toxin (LT), a critical virulence factor of Bacillus anthracis, is a complex of protective antigen (PA) and lethal factor (LF). PA acts to deliver LF to the cytosol of target cell and LF is a Zn2+-dependent metalloprotease, cleaves mitogen activated protein kinase kinases (MAPKKs) family through proteolysis of their N-terminal. Megakaryocyte maturation involves the development of polyploidy cells via endomitosis. Our previous studies have demonstrated that LT can block megakaryocytic polyploidy in TPA (12-O-tetradecanoylphorbol-13-acetate) treated HEL (human erythroleukemia) cell line and found cofilin differentially expressed in TPA and LT pretreatments conditions by 2D-PAGE (two-dimensional polyacrylamide gel electrophoresis) techniqne. Cofilin is a key regulator of actin filament dynamics and reorganization by stimulating depolymerization and severance of actin filament. The activity of cofilin is regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form being inactive. In this study, found the expression level of phospho-cofilin was increased after TPA treatments and reduced to basal level when LT pretreatments by Western blotting. We also demonstrated that regulation of the phospho-cofilin and the upstream LIMK kinase are regulated by ERK/MAPK pathway.
中文摘要................................................................................................................... I
Abstract ................................................................................................................... III
前言...................................................................................................................... - 1 -
一、炭疽桿菌(Bacillus anthracis)...................................................... - 1 -
二、炭疽桿菌(Bacillus anthracis) 的毒力因子................................ - 2 -
三、Lethal toxin (LT) 的標的細胞(target cell) .................................. - 3 -
四、巨核細胞(megakaryocyte) ......................................................... - 4 -
五、Cofilin 與F-actin........................................................................... - 5 -
六、MAPK (mitogen-activated protein kinase) pathway 與cofilin..... - 8 -
實驗目的............................................................................................................ - 10 -
材料與方法........................................................................................................ - 11 -
一、細胞試驗.................................................................................... - 11 -
1. 細胞培養與藥物處理: ............................................................ - 11 -
2. 細胞毒性(cytotoxicity) 試驗: ............................................... - 12 -
3. 多倍體(polyploidy) 分析:..................................................... - 13 -
4. 細胞表面抗原分析: ................................................................ - 13 -
5. 細胞生長動力學測量................................................................ - 14 -
V
6. 利用phalloidin 測定細胞中F-actin 的量:............................. - 15 -
二、蛋白質分析與樣品處理............................................................ - 15 -
1. SDS-PAGE (sodium dodecylsulfate polyacrylamide gel
electrophoresis):...................................................................................... - 15 -
2. Coomassie blue 染色:............................................................. - 16 -
3. 蛋白質濃度測定: .................................................................... - 16 -
4. 西方墨點法(Western blotting): ............................................ - 17 -
5. 細胞溶出物(total cell lysate) 的製備: ................................. - 19 -
6. 細胞核與細胞質蛋白質的分離: ............................................ - 19 -
三、重組蛋白質製備........................................................................ - 21 -
1. 重組質體(plasmid):............................................................... - 21 -
2. 轉化作用(transformation): ................................................... - 21 -
3. 大量蛋白質誘導: .................................................................... - 21 -
四、蛋白質純化(protein purification) [36]:................................. - 22 -
1. 重組致死因子(rLF):............................................................... - 22 -
2. 重組保護性抗原(rPA): .......................................................... - 23 -
結果.................................................................................................................... - 25 -
一、LT 之細胞毒殺實驗.................................................................... - 25 -
二、LT 對細胞分化與生長動力趨勢的影響.................................... - 26 -
VI
1. 多倍體(polyploidy) 分析......................................................... - 26 -
2. 表面抗原(CD41b) 的表現....................................................... - 27 -
3. LT 對HEL 細胞存活的影響........................................................ - 28 -
三、LT 對細胞中p-cofilin 的影響..................................................... - 29 -
四、LT 對F-actin 的影響................................................................... - 31 -
五、MAPK 訊息傳遞路徑對p-cofilin 的影響.................................. - 33 -
六、MAPK 訊息傳遞途徑對LIMK 的影響....................................... - 36 -
七、重組蛋白[rLF、rLF (M)、rPA] 對p-cofilin 的影響................ - 37 -
1. 重組蛋白純化結果: ................................................................ - 37 -
2. 重組蛋白[rLF、rLF (M)、rPA] 之細胞毒殺試驗.................... - 37 -
3. 重組蛋白(rLF、rLF(M)、rPA) 對HEL 多倍體化的影響....... - 38 -
4. 重組蛋白(rLF、rLF(M)、rPA) 對HEL p-cofilin 表現的影響.. - 38 -
討論.................................................................................................................... - 40 -
1. 巨核細胞(megakaryocyte) 多倍體(ploidy) 的生成是因為
p-cofilin 影響F-actin 動態平衡(dynamic) 所造成................................. - 41 -
2. LIMK 1 表現量的上升是p-cofilin 上升的主因......................... - 42 -
3. 巨核細胞表面專一性的抗原(specific antigen) 與多倍體(ploidy)
的生成,是透過不同的訊息傳遞途徑來調控........................................ - 42 -
4. p38 調控p-cofilin 可能不是透過LIMK..................................... - 43 -
VII
5. LT 的活性決定於LF 的catalytic site ......................................... - 44 -
參考文獻............................................................................................................ - 45 -
圖表.................................................................................................................... - 49 -
附圖.................................................................................................................... - 72 -
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