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研究生:宋楚文
研究生(外文):SUNG-CHU WEN
論文名稱:以臨床分離之鮑氏不動桿菌於小鼠模式所產生炎症反應變化之探討
論文名稱(外文):Study on Acinetobacter baumannii isolated from hospital induced inflammatory responses in mice model
指導教授:陳有任
指導教授(外文):Chen, Yu-Jen
學位類別:碩士
校院名稱:元培科技大學
系所名稱:醫學檢驗生物技術研究所
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:99
語文別:中文
論文頁數:69
中文關鍵詞:鮑氏不動桿菌多重抗藥性小鼠腹腔內菌血症
外文關鍵詞:Acinetobacter baumanniimultidrug-resistantmiceintra-peritonealbacteremia
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鮑氏不動桿菌(Acinetobacter baumannii) 為一好氧非葡萄糖發酵革蘭氏陰性球桿菌,廣泛存在於自然的環境,在乾燥環境中可存活長達13天,甚至於超過2星期,此特性使其得以長期存在醫院內乾燥或潮濕的環境中,而成為重要的伺機性致病菌。由於鮑氏不動桿菌很容易產生具抗藥性的新菌株,近年發現許多多重抗藥性菌株,只能藉由多種抗生素合併使用治療。
對於鮑氏不動桿菌的伺機性感染,目前有許多相關研究探討,多數以肺部與傷口感染,然而我們的實驗設計為小鼠動物模式: 從醫院菌血症病患分離出多重抗藥菌株(MDRAB)經培養經由腹腔注射,以109 CFU注射3小時後,抽血做血液抹片染色及細菌培養呈現陽性;犧牲小鼠取部分肝、脾及腎三種器官均質化做細菌培養也呈現陽性反應,顯示早期即產生菌血及可能形成全身性器官感染,其24小時死亡率達100 %。注射低107 CFU,不同時間抽血做抹片染色及細菌培養,在 12小時後有2/3呈現陽性,於24小時後則為陰性;任何時間點取肝、脾及腎三種器官均質化做細菌培養皆呈現陰性,顯示107 CFU需較長時間才會產生菌血但不會形成全身性器官感染,在 24 小時死亡率為 0 %。在小鼠的白血球計數上其結果在注射細菌3小時後,白血球總量下降至2000/mm3 (正常4000-5000/mm3) ,但是在下降白血球總量中之嗜中性球比例卻明顯增加到30%以上,而淋巴球之比例則下降至小於80 % 。另外組織切片染色在109 CFU注射下,肝、脾及腎臟則都有大量嗜中性球浸潤,6-12小時後肝臟與腎臟充血,同時產生肝臟與脾臟膿腫現象。尤其在組織方面某些的變異,是其他老鼠模式動物試驗較少提及。我們也測量細菌注射後3-12小時血液中細胞激素TNF-α以及IL-1β之量的變化,呈現出TNF-α以及IL-1β都有很顯著的增加並與注射菌量呈正向關係。另外發現細菌由腹腔注射後,發現也會導致小鼠腹瀉與鼻腔出血。因此本研究以腹腔注射抗藥性鮑氏不動桿菌,的確可引發菌血,並造成血液白血球量的改變,器官細菌與白血球的浸潤,而細胞激素也明顯的增加。所以我們在此建構的小鼠感染動物模式,有助於此菌感染機制之後續探討和研究。

Acinetobacter baumannii (AB) is an aerobic, non-fermenting, Gram-negative bacillus and distributed widespread in the natural environment, could survival in a dry environment for up to 13 days, even longer than 2 weeks. AB can persistently living in dry or moist hospital environment in this special characteristic become an important opportunistic pathogenic bacteria. AB is easily produced a new antibiotic resistant strain in recent years and many multiple-resistant strains are emerged, and the only treatment method is antibiotics combination therapy. The past researches for opportunistic infection of AB were focused on the infections from lung or wound. However, our experimental design is in mouse model: multi-resistant strains (MDRAB) isolated from the hospital patients. 109 CFU of bacteria are injected by intra-peritoneal, mice were sacrificed after 3 hours then blood smear staining and bacterial cultures were all positive. The bacterial cultures of homogeneous liquids from liver, spleen or kidney were also positive. These results indicated that the production of bacteremia was in early stages to induce systemic infection and the mortality rate reached 100% after 24 hours. The blood smear staining and bacterial culture were two-thirds positive under the injection of 107 CFU after 12 hours, but the results were negative after 24 hours. The bacterial cultures of homogeneous liquids from liver, spleen or kidney were negative in any periods, indicating the inoculation of 107 CFU required longer duration to produce bacteremia, and the death was 0 % after 24 hrs showed would not form the systemic infection. The WBC counts were less than 2000/mm3 (normal 4000-5000/mm3) after 3 hours MDRAB injection. However, the ratios of neutrophils were significantly increased above 30%. The ratio of lymphocytes decreased to below 80% in low concentrations treatment. Usage of tissue biopsy stain, the liver, spleen and kidneys were infiltrated by large number of neutrophils, liver and spleen was congested after 6-12 hours and liver and spleen displayed abscess (abscess) phenomenon under high bacteria concentration treatment. In particular, some of variations for tissue biopsy are not mentioned by other mice model studies. We also measured the production of the cytokine TNF-α and IL-1β from blood after bacteria infection. The productions of TNF-α and IL-1β showed a positive relationship to the dose of injection. However, the productions were beginning decline after injection for 12 hours. The results also showed that multi-drug resistant Acinetobacter baumannii lead to diarrhea and nasal bleeding. Thus, mice injected MDRAB by intraperitoneal actually induced bacteremia, change the amount of WBC in blood. The organs infiltrated by bacteria and WBC. The productions of cytokines were also increased significantly. Therefore, the injection of MDRAB in mice model could benefit for future investigation and research on the infection mechanism.
致謝...................................................................................................................I
中文摘要............................................................................................................III
英文摘要............................................................................................................V
目錄....................................................................................................................VII
表目錄................................................................................................................IX
圖目錄................................................................................................................X
縮寫字................................................................................................................XII
緒論
第一章 背景介紹..............................................................................................1
1.1 鮑氏不動.....................................................................................................1
1.2 發炎反應.....................................................................................................4
1.3 研究目的.....................................................................................................9
第二章 實驗材料與方法………………………………………………………11
1. 鮑氏不動桿菌之處理
1.1 菌株篩選分離……………………………………………………………11
1.2 動物試驗菌液的配製……………………………………………………12
1.3 細菌蠟塊製作……………………………………………………………12
VIII
2.動物處理
2.1 動物之細菌注射………………………………………………………13
2.2 血液分析………………………………………………………………13
2.3 組織切片………………………………………………………………14
2.4染色
2.4.1脫蠟…………………………………………………………………14
2.4.2 H-E 染色(Hematoxylin-eosin stain)……………………15
2.4.3 Gram stain革蘭氏染色………………………………………15
2.4.4 Liu stain劉氏染色……………………………………………15
2.5 閲片…………………………………………………………………16
2.6器官組織均質化處理…………………………………………………16
3. 細胞激素的測定………………………………………………………17
4. 統計分析………………………………………………………………18
第三章實驗結果……………………………………………………………………………19
第四章 討論…………………………………………………………………29
第五章 表……………………………………………………………………36
第六章 圖……………………………………………………………………38
第七章 參考文獻……………………………………………………………59
第八章 附錄…………………………………………………………………65
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