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標題: | 探討細菌第二型拓樸異構酶在氧化逆境下造成的DNA損壞中所扮演之角色 Study on the role of bacterial topoisomerase II in oxidative stress-induced DNA damage |
作者: | Meng-Wen Chung 鍾孟彣 |
指導教授: | 李財坤(Tsai-Kun Li) |
關鍵字: | 拓樸異構酶,氧化逆境,吞噬作用, topoisomerase,oxidative stress,phagocytosis, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 氧化逆境 (oxidative stress) 在細胞層面上扮演許多不同的角色,例如在巨噬細胞 (macrophage) 中進行的殺菌作用 (bactericidal effect) 。然而,活性氧分子 (reactive oxygen species, ROS) 殺菌的機制到目前為止仍不清楚。本篇論文嘗試以過氧化氫 (雙氧水, hydrogen peroxide, H2O2) 代表活性氧分子來探討細菌第二型拓樸異構酶 (Type II topoisomerases, Topo IIs) 在活性氧分子所造成之細菌死亡中所扮演的角色。我們的實驗結果指出過氧化氫可以毒化 (poison) 第二型拓樸異構酶而使之與DNA共價鍵結形成複合體 (cleavable complex) 的形式,這樣的毒化複合體持續存在會導致DNA的斷裂,並進一步造成細菌的死亡。細菌第二型拓樸異構酶在巨噬細胞進行吞噬作用 (phagocytosis) 所造成的殺菌效果中扮演重要角色的推論可由以下結果推得: (1) 過氧化氫可大量活化細菌在遭受DNA損害時的典型SOS反應 (SOS response); (2)過氧化氫可以導致細菌DNA斷裂形成大分子的DNA片段; (3) 這些過氧化氫所造成的大片段DNA具有蛋白質共價連結其上; (4) 這些過氧化氫造成大片段DNA的斷裂反應是可逆的; (5) 細菌第二型拓樸異構酶的抑制劑coumermycin A1,可有效地擷抗過氧化氫所造成的大片段DNA的形成及降低過氧化氫所引起的SOS反應的發生; (6) 最重要的是,細菌第二型拓樸異構酶的抑制劑被證實可以抑制過氧化氫所造成之細菌毒殺作用 (cytotoxicity) 以及巨噬細胞中所進行的吞噬作用之殺菌的效果。總結以上,本篇論文證明了過氧化氫可以有效地藉由毒化細菌第二型拓樸異構酶來造成DNA損壞,並進一步導致細菌的死亡。這個實驗結果提供了一個在吞噬作用中新的殺菌機制:在氧化逆境導致的殺菌作用,特別是在巨噬細胞進行的吞噬作用中,細菌第二型拓樸異構酶扮演了關鍵的角色。 Oxidative stress plays important roles in many cellular states including bactericidal effect during phagocytosis. However, the bacterial killing mechanism by reactive oxygen species (ROS) remains obscure. Here, we investigated the role of bacterial type II topoisomerases (Topo IIs) in oxidative stress-mediated cell death. Hydrogen peroxide (H2O2) was used to induce oxidative stress in bacteria. The involvement of Topo II-mediated DNA damage in bactericidal activity during phagocytosis is supported by the following observations. (1) Cellular exposure of H2O2 led to activation of SOS response, an observation suggestive of DNA damage in E. coli. (2) H2O2 rapidly induced cleavage of nucleoid DNA into high-molecular-weight (HMW) DNA fragments in E. coli. (3) H2O2-induced HMW DNA fragments were covalently linked with proteins. (4) H2O2-induced HMW DNA fragmentations were highly reversible. (5) Coumermycin A1, a bacterial Topo II catalytic inhibitor, efficiently antagonized the formation of H2O2-induced HMW DNA fragments and the H2O2-induced SOS response. (6) Most importantly, coumermycin A1 attenuated the bacterial cell killing by H2O2 and bactericidal effect during phagocytosis in macrophage. Taken together, we concluded that H2O2 effectively targets bacterial Topo IIs and causes DNA breakage, which then leads to cell death. Our results therefore present the first demonstration that bacterial Topo IIs are the bactericidal target during phagocytosis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34292 |
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顯示於系所單位: | 微生物學科所 |
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