臺灣博碩士論文加值系統

群體感應（quorum sensing）系統是為細菌與細菌之間溝通的管道，具有調控整體細菌族群基因表現的功能，亦有研究證實可以調控許多生理功能，如生物膜的形成及毒素的分泌。奈米脫層黏土是為新興的奈米材料，是由蒙托土經過脫層至奈米尺度的奈米矽片（nanoscale silicate platelets，NSP）。研究顯示，NSP可以黏附並包覆菌體及吸附化合物，也可以在NSP接上不同的原子或化合物使其產生附加功能，如奈米矽片銀（AgNSP），即是將銀原子接在NSP上。後續的研究發現，AgNSP採用與銀離子不一樣的殺菌機制，在綠膿桿菌（Pseudomonas aeruginosa，P.A）有相當顯著的抑菌效果，且對於形成生物膜的Pseudomonas aeruginosa也具有減少厚度及殺菌的能力
。本篇論文的目的是想了解是否NSP影響quorum sensing系統，藉此弱化形成生物膜的能力。所以我們採用quorum sensing研究中常使用的菌株哈維氏弧菌（Vibrio harveyi）做為測試目標，以了解NSP是否可以影響quorum sensing的溝通訊號autoinducer（AI，可分為AI-1、AI-2及AI-3）的濃度。實驗顯示，Vibrio harveyi可以隨著AI的濃度不同反映出濃度效應的冷光值及趨勢，我們以此作為NSP是否吸收AI的指標，並觀察到NSP可以降低冷光的釋放。藉由Vibrio harveyi mutants的研究發現， NSP可能可以直接與AI-1或AI-2結合以達到降低冷光的效果。以上的實驗證明NSP具有結合AI而降低菌體內quorum sensing的作用。

Quorum sensing (QS) system is a pathway of cell-cell communication to regulate whole population’s gene expression. QS system can regulate a lot of the physiological functions such as biofilm formation and toxins secretion. Nanoscale silicate platelets (NSP) is a newly developed nano-material that exfoliated from montmorillonite clay. Our previous studies, demonstrate that NSP can adhere to bacteria surface and conjugate with chemical compounds. In this study, we want to know whether NSP have antibacterial function in Pseudomonas aeruginosa biofilm through weakening the capability of Pseudomonas aeruginosa biofilm formation by interfering with the QS system. We applied Vibrio harveyi which is a well-characterize for QS-related study to understand whether NSP can affect the extracellular communication factor of QS system autoinducer (AI, can be classified into AI-1, AI-2 and AI-3). In this study, Vibrio harveyi expresses luciferase of Vibrio harveyi responses to AI concentration in a dose-dependent manner. The experiment system is used for testing whether NSP influenced AI concentration. When Vibrio harveyi was grown in high NSP concentration, NSP reduced luminescence either in the presence of AI-1 or AI-2. This results support the role of NSP to bind AI in culture medium.

摘要 i
(二) Abstract ii
(三) 目次 iii
(四) 圖表目次 iv
(五) 簡介 1
一.奈米脫層黏土（nanoscale silicate platelets，NSP） 1
二.綠膿桿菌（Pseudomonas aeruginosa） 1
三.群體感應（quorum sensing） 2
四.哈維氏弧菌（Vibrio harveyi） 2
五.哈維氏弧菌的群體感應系統 3
(六) 材料及方法 4
一.奈米脫層黏土（nanoscale silicate platelets，NSP） 4
二.菌株及培養液 4
三.Autoinducer bioassay medium及plate之製備 4
四.Marine broth及其plate之製備 5
五.無菌培養基（cell-free medium，CFM）之製備 5
六.以奈米脫層黏土混和的無菌培養液之製備 5
七.哈維氏弧菌與奈米脫層黏土共培養 6
八.低起始濃度培養下冷光變化曲線觀測 6
九.冷光值測量及計算 6
十.菌量O.D值測量 7
(七) 結果 8
一.哈維氏弧菌與奈米脫層黏土共培養下冷光的變化 8
二.低起始濃度培養下冷光變化曲線 9
三.在CFM中低起始濃度培養下冷光變化曲線 9
四.在不同濃度cell-free medium培養下的冷光變化 10
五.在不同濃度NSP-treated CFM培養下冷光變化曲線 11
六.在不同類型AI及濃度NSP-treated CFM培養下冷光變化 12
(八) 討論 13
(九) 結論 16
(十) 參考文獻 17

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