變異鏈球菌(Streptococcus mutans)為主要導致口腔疾病如齲齒、牙周病等主要病因，此菌體能轉換口腔中的醣類，創造酸性的口腔環境，破壞牙齒的結構，並分泌胞外基質，黏附於牙齒表面形成生物膜。胞外基質為常被形容為黏液，主要由水份及高分子物質所組成，功能是使菌體能夠黏附於表面，進而發展成具有完整立體構造的生物膜。
吾人利用濃度稀釋的方式製作了單層菌體試片，並藉由不同培養時間製作了不同成長階段:初附著、小菌落、成熟期的生物膜試片，透過原子力顯微術的峰力定量奈米機械模式(PeakForce quantitative nanomechanical property mapping, PeakForce QNM) 觀測大範圍10μm ×10μm生物膜的機械性質分佈，並且鎖定單一菌體約1μm ×1μm的範圍進行追蹤，利用黏滯性、能量耗散、彈性係數變化，分析胞外基質分泌的動態過程。另外，吾人也使用了共軛焦顯微鏡觀測胞外多醣體分佈，並與原子力顯微鏡觀測到的生物膜機械性質分佈交互對照。
實驗討論主要分為兩部份，在大範圍生物膜機械性的觀測中，吾人討論了三種成長階段的能量耗散與黏滯性變化，發現於小菌落階段的黏滯性及能量耗散數值最高，其次為成熟期，初附著則數值最低，並發現此量測結果與胞外多醣體的分泌量成正相關。於單一菌體胞外基質的追蹤觀測中，吾人主要觀測菌體周圍黏滯性、彈性係數的變化。在初附著階段中，胞外基質黏滯性及彈性係數變化速度與該位置和菌體的距離相關，推測為胞外基質分泌時，所需要的分子物質運送至該處由距離造成的時間長短所致。在成熟期的生物膜觀測中，菌體周圍胞外基質的黏滯性由內而外數值漸增，將相同黏滯性數值範圍所涵蓋的面積加以統計後，呈現了波動上升的曲線，反應了胞外基質生成及消耗的過程。另外，不論在初附著或是成熟期的生物膜，皆可觀測到於高度落差大的區域優先分泌黏滯性高且彈性係數大的胞外基質，推測為由高度落差產生的剪應力啟動了菌體分泌胞外基質的機制所致。
在本實驗中，吾人成功了利用微區機械性質變化，觀測胞外基質動態分泌過程，於未來的研究中，吾人希望能將此種分析方式與醫學上，與探討分子、基因、蛋白質對生物膜影響的儀器做結合，探討分子物質對於生物膜機械性質分佈的影響，並希望能找出影響生物膜結構的因子與控制方法的，進一步用於醫學治療。

Our main goal on this project is to observe the dynamic extracellular polymeric substance (EPS) secretion process during Streptococcus mutans’ biofilm formation. By Peakforce quantitative nanomechanical property mapping mode (Peakforce QNM) of Atomic force microscopy we obtained the mechanical property data which changed with EPS secretion. From our result, we found that adhesion force and dissipation energy distribution is related to the amount of expolysaccharides accumulation, adhesion force has the highest value at the place with larger height difference and each EPS layer with certain adhesion range are accompanied by EPS formation and consumption.

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