臺灣博碩士論文加值系統

銀奈米粒子 (AgNPs) 因為具有廣譜抗菌的功效而有望成為傳統抗菌劑的替代品。 本實驗室先前以微波消化法將植物萃取物魚腥草 (HCE) 及苦瓜五號 (BG5) 作為還原劑和封端劑合成銀奈米粒子。經殺菌效力分析後顯示魚腥草銀奈米粒子 (HCE-AgNPs) 和苦瓜五號銀奈米粒子(BG5-AgNPs) 對大部分受測菌株具有出色的殺菌活性。然而目前對於細菌是否也容易經由銀奈米粒子誘導而產生抗性及其背後可能機制之研究仍然有限。因此在本研究中，我們首先嘗試通過 AgNPs 誘導細菌產生耐藥性，並進一步進行蛋白質體學分析，以找出可能的機制。我們的實驗結果顯示，部分細菌對這些 AgNPs 的最低抑菌濃度 (MIC) 經誘導後增加了一倍以上。 蛋白質體學分析結果發現，經過這些AgNPs誘導後分別在大腸桿菌和綠膿桿菌中有 1,144 種，及 896 種蛋白表達量有顯著改變。 此外，基因本體論 (Gene Ontology) 分析表明，這些蛋白質的功能與銀、銅離子接觸反應、細菌趨化性、第四型菌毛合成及芳香族氨基酸分解代謝具有較顯著的相關性。我們接著在BG5-E.coli的組別中挑選與銀奈米抗性相關的可能基因cusR、cpxA和cpxR，進行基因重組及表達；發現只有過度表達CusR會對銀奈米粒子的抗性造成影響，而過度表達CpxA和CpxR的生物膜較不易被銀奈米粒子清除。
此外，我們嘗試將HCE-AgNPs以及抗菌肽 (AYG25、AYG 30)進行合併使用，結果發現有加成的效果。因此我們嘗試將帶負電的HCE-AgNPs和帶正電的AYG25、AYG 30混合，讓他們自發性的形成銀奈米粒子及抗菌肽複合物 (AYG-HCE)。實驗結果發現，當抗菌劑濃度AYG30：HCE = 1：2時，能降低最低抑菌濃度，且銀奈米粒子負載抗菌肽之效果最好，而且表現出較佳的血漿抗性與較低的溶血活性。因此我們推測HCE-AgNPs與AYG30形成複合物後，能夠具有較好的抗菌效果及較高的生物相容性。

Silver nanoparticles (AgNPs) are promising alternatives to conventional antimicrobials owing to their efficacy against a wide spectrum of bacteria. In our laboratory, we previously synthesized silver nanoparticles (AgNPs) by the microwave irradiation method, using plant extracts, Houttuynia cordata extract (HCE), and Bitter gourd No. 5 extract (BG5) as reducing and capping agents. Evaluation of the antibacterial efficacy of the HCE-AgNPs and BG5-AgNPs showed excellent bactericidal activity against most tested bacterial strains. However, the studies regarding bacterial resistance and their mechanisms against AgNPs still remains limited and inconclusive. Therefore, in this study, we tried to induce bacterial resistance by AgNPs and further performed proteomic analysis to find out potential mechanisms. Our results indicated that some bacteria's minimum inhibitory concentration (MIC) of these AgNPs was twice more than usual after induction. Proteomic analysis showed that exposure to AgNPs significantly altered the expression of 1,144 and 896 proteins in E. coli and P. aeruginosa, respectively. Additionally, Gene Ontology analysis reveals a significant correlation between the functions of these proteins and their interaction with silver and copper ions, bacterial chemotaxis, type IV pilus synthesis, and aromatic amino acid degradation metabolism. In BG5-E.coli group, the potential genes cusR, cpxA, and cpxR, which are associated with AgNPs resistance, were selected for gene recombination and expression. We found that only overexpression of CusR affected the resistance of AgNPs, while expressing CpxA and CpxR biofilm was less susceptible to silver nanoparticle eradication.
In addition, We attempted to combine HCE-AgNPs and antimicrobial peptides (AYG25, AYG30) and observed an additive effect. Therefore, we tried to spontaneously form a complex (AYG-HCE) by mixing negatively charged HCE-AgNPs with positively charged AYG25 and AYG30. Experimental results showed that when the concentration of antimicrobial agent was at a ratio of AYG30:HCE = 1:2, it could reduce the minimum inhibitory concentration (MIC) and exhibited the best silver nanoparticle loading effect with antimicrobial peptides. Moreover, it demonstrated better bactericidal activity in plasma and lower hemolytic activity. Therefore, we speculate that after the formation of the complex between HCE-AgNPs and AYG30, it could exhibit improved antibacterial efficacy and higher biocompatibility.

摘要 I
Abstract III
第一章 前言 1
1.1 抗生素的耐藥性 1
1.2 銀奈米粒子 2
1.3 奈米材料的汙染 3
1.4 細菌對金屬奈米粒子的耐藥機制 4
1.5 抗菌肽 4
1.6 聯合治療 (Combined therapy) 6
1.7 銀奈米粒子與抗菌肽的交互作用 6
1.8 研究目的 7
第二章 實驗材料 8
2.1 菌種 8
2.2 培養基 9
2.3 引子 10
2.4 限制酶 12
2.5 抗生素 12
2.6 質體 12
2.7 藥品與試劑 13
2.8 實驗套件 (Experiment kits) 15
第三章 研究方法 16
3.1 植物萃取液合成銀奈米粒子 16
3.2 最低抑菌濃度測試 (Minimum inhibitory concentration) 16
3.3 抗菌劑適應現象 (Evolutionary adaptation to antimicrobials) 17
3.3.1 生長曲線 (Growth rate) 17
3.3.2 最低生物膜根除濃度 (Minimum biofilm eradication concentration) 18
3.4 蛋白質體學分析 (Proteomics analysis) 18
3.4.1 LC-MS 檢測 18
3.4.2 蛋白的鑑定和數據分析 20
3.5 即時定量聚合酶連鎖反應 (Real Time - Polymerase Chain Reaction) 20
3.5.1 純化total RNA 20
3.5.2 反轉錄聚合酶連鎖反應 (Reverse Transcription - Polymerase Chain Reaction) 21
3.5.3 即時定量聚合酶連鎖反應 (Real Time - Polymerase Chain Reaction) 22
3.6 基因功能分析 (Gene function) 23
3.6.1 細菌DNA的萃取 23
3.6.2 TA選殖 (TA cloning) 23
3.6.3 質體純化 24
3.6.4 基因選殖 25
3.6.5 基因表達 26
3.7 以抗菌肽修飾銀奈米粒子 26
3.7.1 抗菌肽設計 26
3.7.2 通過棋盤分析進行協同作用測量 (Synergy checkerboard assay) 27
3.7.3 抗菌肽銀奈米粒子的合成 28
3.7.4 溶血試驗 (Haemolysis assay) 29
3.7.5 血漿抗性試驗 29
3.7.6 穿透式電子顯微鏡Transmission electron microscopy (TEM) 30
3.7.7 AYG30-HCE於不同溶液中的穩定性 30
3.8 統計分析 30
第四章 研究結果 32
4.1 銀奈米粒子的體外抗菌試驗 32
4.2 細菌連續傳代對抗菌劑產生的適應現象 32
4.3 傳代菌株對銀奈米粒子產生抗性 33
4.4 連續傳代菌株的生長曲線 35
4.5 對銀奈米粒子產生抗性的菌株蛋白質體學分析 35
4.6 差異表達蛋白的基因功能分析 36
4.7 調控奈米粒子抗性的相關基因 38
4.8 標的蛋白表達增加對銀奈米粒子抗性的影響 39
4.9 新型抗菌劑之間的協同作用 41
4.10 AYG30-HCE複合物的生物相容性 42
4.11 穿透式電子顯微鏡結果 43
4.12 AYG30-HCE於不同溶液中的穩定性 43
第五章 結論與討論 45
第六章 圖片與表格 52
圖一、 細菌連續傳代對抗菌劑產生的適應現象 52
圖二、 連續傳代菌株的最低抑菌濃度變化 53
圖三、 連續傳代的抗性菌株之生長曲線 54
圖四、 連續傳代未具抗性菌株之生長曲線 55
圖五、 BG5-E. coli差異表達蛋白的比較 56
圖六、 HCE- P. aeruginosa差異表達蛋白的比較 57
圖七、 差異表達基因的基因功能分析 58
圖八、 BG5-E.coli 蛋白質相互作用網路圖 59
圖九、 BG5-E.coli蛋白質組的基因進行富集分析 61
圖十、 HCE- P. aeruginosa 蛋白質相互作用網路圖 62
圖十一、 HCE- P. aeruginosa蛋白質組的基因進行富集分析 63
圖十二、 差異表達基因即時定量分析 64
圖十三、 建構表達目標蛋白之質體流程圖 65
圖十四、 誘導表達之目標蛋白 66
圖十五、 標的蛋白表達增加對生長速率的影響 67
圖十六、 生物膜的差異表達 68
圖十七、 抗菌劑表徵與生物相容性 69
圖十八、 TEM成像 70
圖十九、 抗菌劑於不同溶液中的穩定性 71
圖二十、 抗菌肽-奈米粒子複合物的殺菌機制模擬示意圖 72
表一、 銀奈米粒子體外抗菌試驗 73
表二、 經AgNPs誘導出之抗性菌株的標的基因表達分析結果 74
表三、 大腸桿菌標的基因過度表達後對銀奈米粒子之MIC影響 75
表四、 大腸桿菌對銀奈米粒子產生抗性後的突變位點 76
表五、 抗菌劑協同作用 77
表六、 AYG-HCE複合物的MIC及負載效果 78
附檔一、 許可協議 79
第七章 參考文獻 80

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