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研究生:林姿吟
研究生(外文):Tzu-Yin Lin
論文名稱:探討金奈米粒子於化膿性鏈球菌感染中之保護角色
論文名稱(外文):The protective role of Au nanoparticles in Streptococcus pyogenes infection
指導教授:蔡佩珍蔡佩珍引用關係張凱誌
指導教授(外文):Pei-Jane TsaiKai-Chi Chang
學位類別:碩士
校院名稱:慈濟大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:60
中文關鍵詞:化膿性鏈球菌金奈米粒子近紅外線光源
外文關鍵詞:Streptococcus pyogenesAu nanoparticlesnear-infraredlight (NIR)
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化膿性鏈球菌為臨床上常見的感染性致病菌,造成化膿性、非化膿性或嚴重侵入性感染疾病。然而抗菌的治療使得抗藥性菌株增加,因此發展有別於傳統的新興治療方式是迫切需要的。最新研究指出,金奈米粒子具有吸收近紅外光的能力,廣泛應用於臨床治療上。過去研究已於體外試驗中證實,於近紅外光源激發後金奈米粒子可以將光能轉換成熱能,對細菌造成不可逆的傷害。因此,我們想進一步證實在動物試驗中,近紅外光激發金奈米粒子誘發的高溫於化膿性鏈球菌感染上是否提供保護的效果。首先,我們發現當溫度高於46℃ 即可抑制化膿性鏈球菌的生長。體外試驗結果顯示近紅外光激發金奈米粒子誘發的高溫確實可以降低細菌數量。為了模擬真實皮下感染系統,我們將皮膚平鋪在化膿性鏈球菌與近紅外線光源之間,同時發現金奈米粒子激發產熱後會抑制細菌的生長,並且隨著照射時間與金奈米粒子增加細菌量隨之減低,顯示金奈米粒子的抑菌功能並不會被皮膚覆蓋所影響。由細菌清除率實驗結果證實,給予金奈米粒子治療後能有效降低局部感染部位之細菌數;在細菌量降低的情況下也降低發炎反應相關之細胞激素 (IL-6) 與趨化激素 (KC, MCP-1) 的mRNA的表現量。由於金奈米粒子具有良好的殺菌能力,因此於致死率實驗發現給予金奈米粒子治療後可以增加感染小鼠之存活率。綜合研究結果,顯示金奈米粒子透過降低化膿性鏈球菌之感染菌量與發炎反應,進而增加感染小鼠之存活率,達到化膿性鏈球菌感染的保護效果。
Streptococcus pyogenes (S. pyogenes) is a clinical pathogen that commonly causes suppurative, non-suppurative and severe invasive infections. The standard treatment of S. pyogenes infection is antimicrobial agent which, however, increases resistant strains. Thus, developing new therapeutic methods and tools distinct from traditional strategies is urgent. Au nanoparticle that has unique optical properties can be used as a photothermal agent in the clinical therapy. Our previous study demonstrated that, under near-infrared (NIR) light irradiation, Au nanoparticles rapidly converted light to heat and subsequently destroyed the bacteria in vitro. Thus, we hypothesized that hyperthermia induced by NIR-irradiated Au nanoparticles protects from streptococcal infection in animals. First, we found that S. pyogenes growth was inhibited when the temperature was higher than 46°C. Hyperthermia induced by NIR-irradiated Au nanoparticles significantly reduced bacterial numbers in vitro. To mimic the in vivo subcutaneous infection model, we placed mouse skin between bacterial suspension and NIR light source. We found that NIR-irradiated Au nanoparticles decreased bacterial growth in a dose- and time-dependent manner, suggesting the bactericidal effect of Au nanoparticles can be achieved in spite of appearance of a physical barrier. The in vivo bacterial clearance experiment showed that the bacterial numbers in local exudates from S. pyogenes-infected mice with treatment of NIR-irradiated Au nanoparticles were significantly decreased. Moreover, the decreased bacterial numbers in mouse skin were associated with reduced mRNA levels of proinflammatory cytokines (IL-6) and chemokines (KC, MCP-1). The mortality assay showed that NIR-irradiated Au nanoparticles significantly increased the survival rate of S. pyogenes-infected mice. Taken together, treatment of NIR-irradiated Au nanoparticles reduced the bacterial numbers and systemic inflammatory responses to provide the protection from and host survival on infection of S. pyogenes.
致謝..................................................................................................... I
中文摘要............................................................................................. IV
英文摘要............................................................................................. VI
目錄..................................................................................................... VIII
表目錄................................................................................................. XII
圖目錄................................................................................................. XIII
第一章、緒論....................................................................................... 1
第二章、材料與方法........................................................................... 9
2-1 材料............................................................................................. 9
2-1-1 金奈米粒子 (Au nanoparticles).............................................. 9
2-1-2 近紅外線光源 (Near-infrared light, NIR).............................. 9
2-1-3 細菌株 (Bacteria strain).......................................................... 9
2-1-4 實驗動物 (Animals) ............................................................... 9
2-2 方法............................................................................................. 10
2-2-1 體外試驗 (in vitro).................................................................. 10
2-2-1-1 細菌培養與製備................................................................... 10
2-2-1-2 溫度抵抗性試驗................................................................... 11
2-2-1-3 近紅外線光源激發金奈米粒子之光熱殺菌測試............... 11
2-2-1-4 模擬真實皮下感染模式與殺菌試驗................................... 12
2-2-2 活體動物試驗 (in vivo).......................................................... 13
2-2-2-1 麻醉藥劑 (Avertin) 之配製................................................ 13
2-2-2-2 小鼠細菌感染模式 (mice infection model)........................ 13
2-2-2-3 小鼠皮下感染後之清除率試驗 (Clearance)...................... 14
2-2-2-4 感染小鼠之存活率觀察 (Survival rate)............................. 14
2-2-2-5 活體動物之生物毒性測試................................................... 14
2-2-2-6 組織RNA萃取 (RNA extraction)....................................... 14
2-2-2-7 反轉錄聚合�○s鎖反應 (Reverse transcriptase-PCR)....... 15
2-2-2-8 即時聚合�○s鎖反應 (Real-time PCR, Q-PCR)................ 16
2-2-2-9 血清生化數值分析............................................................... 17
2-2-3 統計分析法與繪圖軟體.......................................................... 17
第三章、結果..................................................................................... 18
3-1 於體外試驗中探討金奈米粒子清除化膿性鏈球菌之效力..... 18
3-1-1 探討化膿性鏈球菌對溫度的抵抗能力.................................. 18
3-1-2 探討金奈米粒子清除化膿性鏈球菌之效力.......................... 19
3-1-3 近紅外線光源與小鼠間之安全照射距離.............................. 20
3-1-4 於近紅外線光源之安全距離下探討金奈米粒子清除化膿
性鏈球菌的效力......................................................21
3-2 於體外試驗模擬皮下感染模式中探討金奈米粒子清除化膿
性鏈球菌之效力......................................................22
3-2-1 探討金奈米粒子清除化膿性鏈球菌之效力.......................... 22
3-2-2 探討金奈米粒子濃度對清除化膿性鏈球菌之效力影響...... 24
3-3 利用動物感染模式觀察金奈米粒子對於化膿性鏈球菌生長
的抑制效果與宿主保護角色..............................................................25
3-3-1 於短時效下測試金奈米粒子對於化膿性鏈球菌生長之
抑制效果.........................................................................25
3-3-2 於長時效下測試金奈米粒子對於化膿性鏈球菌生長之
抑制效果..................................................................26
3-3-3 探討金奈米粒子對於宿主之保護機制.................................. 28
3-3-3-1 金奈米粒子治療下血液生化質之變化............................... 28
3-3-3-2 局部感染患部之細胞發炎激素和趨化激素的表現量....... 29
3-4 利用動物感染模式分析金奈米粒子對於化膿性鏈球菌感染
小鼠之存活率影響.....................................................................
31
3-5 於活體試驗中探討金奈米粒子對於宿主的毒性傷.............. 31
第四章、討論....................................................................................... 33
參考文獻............................................................................................. 39
表......................................................................................................... 44
圖......................................................................................................... 46
附錄..................................................................................................... 58
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