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研究生:邱健昭
論文名稱:綠膿桿菌外毒素A引起大鼠肝毒性模式之建立
論文名稱(外文):Pseudomonas aeruginosa exotoxin A induced hepatotoxicity:an animal model in rats
指導教授:陳三多
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫病理學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:外毒素A肝毒性細胞凋亡
外文關鍵詞:Exotoxin AHepatotoxicityApoptosis
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綠膿桿菌為一種機緣性感染菌,本菌在感染時會分泌外毒素(Pseudomonas aeruginosa exotoxin A, PEA)及porin等多種毒力物質。PEA可引起實驗動物及人之肝毒性,基於此,本實驗致力於PEA引起大鼠肝毒性之機制研究,並建立大鼠之動物模式。實驗採用雄性六至七週齡SPF Wistar大鼠(每組三至五隻),靜脈注射0(對照組)、10、20、30、40及60μg/kg PEA,並在攻毒後12、24、36、48、60及72小時後犧牲動物。使用臨床觀察、組織病理切片、免疫化學染色法、TUNEL染色、DNA斷片電泳法、偵測血清生化(ALT, AST與黃疸指數)、偵測細胞素及免疫球蛋白等技術分析。結果,在40、60μg/kg劑量下,組織切片觀察中,攻毒後12小時即可看到多數CLAB出現,及引起門脈區及中心靜脈區壞死,且延伸為多發局部性壞死灶或大區域壞死灶,動物並分別在36及24小時內死亡。在20與部分30 μg/kg劑量下雖可造成肝毒性但動物卻可恢復,並在攻毒後60小時後可見ALT及AST下降,及分別於48及60小時可見肝細胞有絲分裂,呈現再生現象。在DNA斷片電泳法方面,在攻毒24小時後分別在30、40、60μg/kg劑量下可見明顯DNA Ladder,而20μg/kg以下劑量組則無此現象。而TUNEL染色則在20μg/kg劑量下,攻毒12小時後即可染出肝細胞發生凋亡,並隨著時間延長及劑量增加而更加嚴重。在10μg/kg劑量下各攻毒時間點,經由以上各種分析與對照組比較均無明顯變化。在細胞素測定方面,則發現在攻毒24小時40 μg/kg試驗組其血清中TNF-α的濃度有極顯著的上升,在24小時下30及40 μg/kg試驗組其IL-2的濃度有顯著的上升,而IL-6在60 μg/kg試驗組於攻毒後12小時有極顯著的上升、IL-10在30 μg/kg試驗組於攻毒後24小時開始上升至48小時為最高峰,而IFN-γ則只有在60μg/kg劑量組有少量出現。另外,免疫球蛋白IgM則在給予30μg/kg於攻毒48小時後大幅揚升,顯示動物如耐過PEA攻擊即可產生大量之IgM。本實驗結果發現由以上各種肝毒性評估方法中,免疫組織化學染色最為敏感,而其他方法可作為輔助診斷。並且由細胞素測定顯示其引起大鼠肝毒性與免疫細胞過度活化有關。由建立PEA引起大鼠之肝毒性模式,將來可用於致病機制與不同藥物對PEA引起肝毒性保護能力之探討。

目錄
誌謝
目錄………………………………………………………………………I
表次及圖次………………………………………………………………III
中文摘要…………………………………………………………………1
第一章 文獻探討…………………………………………………………3
1.外毒素A之背景…………………………………………………………3
2.外毒素A與肝毒性之關係………………………………………………6
3.外毒素A與其它組織細胞之關係………………………………………9
4.外毒素A與免疫之關係…………………………………………………11
5.外毒素A於生物醫學之應用……………………………………………16
第二章 研究動機與目的…………………………………………………18
第三章 材料與方法……………………………………………………20
1.試驗材料………………………………………………………………20
2.試驗方法及步驟………………………………………………………26
第四章 結果………………………………………………………………32
1.臨床觀察………………………………………………………………32
2.肉眼病變………………………………………………………………32
3.組織病理………………………………………………………………33
4.組織化學染色…………………………………………………………35
5.TUNEL染色………………………………………………………………36
6.DNA斷片電泳法…………………………………………………………36
7.血清生化………………………………………………………………37
8.細胞素分析……………………………………………………………38
9.免疫球蛋白分析………………………………………………………40
第五章 討論………………………………………………………………41
1.大鼠與小鼠致死劑量上之不同………………………………………41
2.偵測肝細胞凋亡方法之比較…………………………………………42
3.偵測肝細胞壞死方法之評估…………………………………………43
4.肝內CLAB之探討………………………………………………………44
5.PEA對其他組織細胞傷害之探討………………………………………45
6.PEA引起細胞素變化之探討……………………………………………46
7.免疫球蛋白之探討………………………………………………………49
第六章 結論………………………………………………………………51
英文摘要……………………………………………………………………53
參考文獻……………………………………………………………………69

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