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研究生:包翊絹
研究生(外文):I-Juian Bau
論文名稱:奈米銀脫層黏土之抗菌機制及應用
論文名稱(外文):Antimicrobial mechanism and applications of silver nanoparticles on the exfoliated platelet clay
指導教授:蘇鴻麟蘇鴻麟引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:53
中文關鍵詞:奈米銀抗菌機制掃描式電子顯微鏡金黃色葡萄球菌大腸桿菌沙門氏桿菌鮑氏不動桿菌
外文關鍵詞:silver nanoparticlesantimicrobial mechanismScanning Electron MicroscopeStaphylococcus aureusEscherichia coliSalmonella typhimuriumAcinetobacter baumannii
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銀或含銀化合物能抗菌力,而經奈米化的銀粒子也具有殺菌的功效,但其抗菌機制目前尚不清楚。本研究使用的奈米銀黏土材料AgNP/NSP與AgNP/SWN是使用NSP脫層黏土及SWN蒙脫土作為分散劑,以防止奈米銀粒子在製程中產生聚集,藉以研究奈米銀的抗菌機制。首先利用AgNP/NSP與AgNP/SWN對具多重抗藥性菌株鮑氏不動桿菌(Acinetobacter baumannii)及具有銀離子抗性之大腸桿菌菌株( E. coli strain J53 pMG101)作抑菌能力分析並與硝酸銀的抗菌能力作比較,比較後的結果顯示在相同銀濃度下, AgNP/NSP對此兩種菌均具有最好的抑菌能力。以掃瞄式電子顯微鏡(Scanning Electron Microscope)觀察經奈米銀黏土處理的細菌發現材料將其表面包覆,且使細菌聚集。加入propidium iodide (PI)後24小時,吸附於奈米銀黏土上的細菌內的DNA並無呈現紅色螢光,證明細菌的細胞膜大致完好,仍可抵抗PI進入細菌內,但是否因細胞膜仍有輕微損害而導致抑制細菌生長仍不清楚。使用大腸桿菌E. coli strain ML-35p,觀察到奈米銀黏土的確會增加細菌細胞膜的通透性,因此推測奈米銀黏土材料藉由此方式影響細菌的分裂而達到殺菌的功效。也發現只有高於0.1wt%奈米銀黏土可顯著降低β-galactosidase的酵素活性,證明奈米銀導致蛋白質變性的能力並不強。除細菌外,也發現AgNP/NSP與AgNP/SWN具有抑制真菌生長之特性並會將真菌表面包覆。另外在奈米銀脫層黏土材料應用於治療家禽細菌性下痢以取代抗生素的實驗中,顯示當雛雞感染沙門氏桿菌後的十八小時內,經口投與AgNP/NSP,可能可以減緩細菌經血液蔓延至其他器官而達到治療的效果。使用轉移子 ( transposon ) 對奈米銀粒子抗性基因進行篩選,也未發現突變細菌株,此結果顯示奈米銀並無基因毒性。最後在奈米銀黏土材料的致突變性試驗 (Ames test )中,結果顯示在0.5mg/plate 劑量以下不具有致突變的作用。而未來將更深入探討奈米銀黏土的抑菌機制並將其應用於細菌感染的治療上。
Silver or silver compound can inhibit bacteria growth. Silver nanoparticle also has the bactericidal effect but the antibacterial mechanism is still unknown. AgNP/NSP and AgNP/SWN, the materials used in this article, are synthesized by using nano silicate platelet clays and artificial organic clays as dispersing to stabilize and immobilize silver nanoparticles and consequently used to study the antibacterial mechanism of silver nanoparticles. Firstly, the growth inhibition capability of AgNP/NSP and AgNP/SWN to multidrug-resistant Acinetobacter baumannii and silver ion-resistant E. coli strain J53 pMG101 were analyzed and compared with silver nitrate. The result showed that among these three silver compound AgNP/NSP has the best bactericidal effect under the same silver concentration.Observation by using Scanning Electron Microscope(SEM) revealed that AgNP/NSP encapsulated the bacteria and form multiple clay-cells aggregates. Absorbed bacteria on AgNP/NSP repelled the entry of propiodium iodide(PI) indicating that bacteria cell membrane was not ruptured. But whether the mild lesion of cell membrane leads the growth retardation is still unknown. By using E. coli strain ML-35p, we found that Ag nanoparticles clay indeed could increase the membrane permeability.Therefore we speculate that Ag nanoparticles clay has bactericidal effect through interfering intracellular homeostasis. We also find only higher than 0.1wt% Ag nanoparticles clay can obviously decrease β-galactosidase activity and this result demonstrated the oxidization of functional proteins by Ag nanoparticles was not strong. Besides bacteria , AgNP/NSP and AgNP/SWN also could inhibit the growth of infectious fungi, including Trichophyton rubrum、Trichophyton fluviomuniense、Trichophyton mentagrophytes、Microsporum canis and Microsporum gypseum , the common pathogens of Tinea pedis. For the substitution of antibiotics, Ag nanoparticles clay was applied to cure fowl diarrhea after infection with salmonella. After eighteen hours, AgNP/NSP via oral route retarded bacteria spread to other organ. The Ag nanoparticles- resistant bacteria was not found, showing the safety of clinical application. We further demonstrated that geno-safety of AgNP/NSP by Ames test, showing lower than 0.5mg/plate dose have no genetic mutation. In the future , we will further explore the mechanism of bactericidal effect of AgNP/NSP and apply Ag nanoparticles clay to clinical treatment of bacterial infection.
目次………………………………………………………i
圖表目次…………………………………………………ii
中文摘要…………………………………………………v
英文摘要…………………………………………………vii
前言………………………………………………………1
材料與方法………………………………………………7
結果………………………………………………………27
討論………………………………………………………41
參考文獻…………………………………………………50
結果圖表…………………………………………………54
附錄………………………………………………………86









圖表目次

圖1.奈米矽片結構……………………………………………………………………54
圖2.利用黏土分散高濃度奈米銀粒子的AgNP/NSP 與AgNP/SWN 之結構……55
圖3.奈米銀抗性基因進行篩選實驗過程示意圖……………………………………56
圖4.使用不同濃度AgNP/NSP、AgNP/SWN及AgNO3對具銀離子抗性之大腸桿
菌菌株E. coli J53pMG101進行抑制生長之測試結果…………………………57
圖5.使用不同濃度AgNP/NSP、AgNP/SWN及AgNO3對鮑氏不動桿菌分離株進行
抑制生長之測試結果……………………………………………………………58
圖6.使用不同濃度AgNP/NSP對大腸桿菌突變菌株E. coli △ompA、△ompT、
△nlpІ,△papA、△K1 capsule 進行抑制生長之測試結果……………………59
圖7.利用掃描式電子顯微鏡 ( Scanning Electron Microscope) 觀察奈米銀脫層黏土
AgNP/NSP對金黃色葡萄球菌 (Staphylococcus aureus ) 的影響……………60
圖8.利用掃描式電子顯微鏡 ( Scanning Electron Microscope) 觀察奈米銀脫層黏土
AgNP/NSP對沙門氏桿菌(salmonella typhimurium ) 的影響…………………61
圖9.利用掃描式電子顯微鏡 ( Scanning Electron Microscope) 觀察奈米銀脫層黏土
AgNP/NSP對真菌Trichophyton mentagrophytes的影響………………………62
圖10.利用掃描式電子顯微鏡 ( Scanning Electron Microscope)觀察奈米銀脫層黏
AgNP/NSP對真菌Trichophyton fluviomuniense的影響………………………63
圖11.利用掃描式電子顯微鏡 ( Scanning Electron Microscope)觀察奈米銀脫層黏
AgNP/NSP對真菌Microsporum canis的影響…………………………………64
圖12.利用螢光顯微鏡觀察奈米銀脫層黏土AgNP/NSP對大腸桿菌E.coliDH5α的
影響 ……………………………………………………………………………65
圖13. AgNP/NSP及AgNP/SWN及AgNO3對細菌細胞膜通透性的影響 …………66
圖14.不同濃度的AgNP/NSP、AgNP/SW與黏土NSP、SWN對β-galactosidas活性的影響…………………………………………………………………………67
圖15.奈米銀黏土材料AgNP/NSP與黏土NSP抑制真菌菌絲生長之測試結果……68
圖16.奈米銀黏土材料AgNP/NSP與黏土NSP抑制真菌菌絲生長測試結果量化圖
…………………………………………………………………………………69
圖17.奈米銀黏土材料AgNP/SWN與黏土SWN抑制真菌菌絲生長測試結果量化
圖………………………………………………………………………………70
圖18.奈米銀黏土材料AgNP/NSP、AgNP/SWN與黏土NSP、SWN抑制真菌孢子
生長之測試結果………………………………………………………………71
圖19.奈米銀黏土材料AgNP/NSP與黏土NSP抑制真菌孢子生長測試結果量化圖………………………………………………………………………………72
圖20.奈米銀黏土材料AgNP/SWN與黏土SWN抑制真菌孢子生長測試結果量化
圖………………………………………………………………………………73
圖21.利用奈米銀脫層黏土AgNP/NSP取代抗生素以治療家禽細菌性下痢第一次實
驗結果…………………………………………………………………………74
圖22.利用奈米銀脫層黏土AgNP/NSP取代抗生素以治療家禽細菌性下痢第二次
實驗結果………………………………………………………………………75
表1.致突變性試驗中各菌株所使用致癌劑所需劑量範圍…………………………76
表2.致突變試驗各菌株呈陰性反應之回復突變菌落數範圍………………………76
表3.奈米銀抗性基因篩選結果 ………………………………………………………77
表4.~表6.利用奈米銀脫層黏土AgNP/NSP取代抗生素以治療家禽細菌性下痢實驗
結果………………………………………………………………………78
表7.利用奈米銀脫層黏土取代抗生素以治療家禽細菌性下痢實驗結果統計……79
表8.AgNP/NSP之毒性試驗結果……………………………………………………80
表9.AgNP/SWN之毒性試驗結果……………………………………………………80
表10.NSP之毒性試驗結果……………………………………………………………81
表11.SWN之毒性試驗結果…………………………………………………………81
表12.AgNP/NSP之致突變性試驗結果………………………………………………82
表13.AgNP/SWN之致突變性試驗結果……………………………………………83
表14.NSP之致突變性試驗結果………………………………………………………84
表15.SWN之致突變性試驗結果……………………………………………………85
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