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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王錦堂 | |
dc.contributor.author | Po-An Su | en |
dc.contributor.author | 蘇柏安 | zh_TW |
dc.date.accessioned | 2021-06-16T08:04:03Z | - |
dc.date.available | 2019-10-09 | |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-07-02 | |
dc.identifier.citation | 1. Ryan, K. R., CG, eds. (2004) Sherris Medical Microbiology(4th ed.). McGraw Hill. ISBN 0-8385-8529-9.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/57972 | - |
dc.description.abstract | 近年來,克雷伯氏肺炎桿菌造成的社區侵襲性原發性肝膿瘍成為了全球性的新興感染症。克雷伯氏肺炎桿菌菌體外的莢膜是重要的致病因子之一,可以抵抗宿主免疫細胞吞噬作用以及補體毒殺機制,而在台灣造成原發性肝膿瘍以K1、K2莢膜血清型最為常見,充分顯示莢膜型和致病力的相關性。克雷伯氏肺炎桿菌菌株的多重抗藥性問題日趨嚴重,例如Extended-spectrumβ-lactamase(ESBL)基因及Carbapenem-resistant Klebsiella pneumoniae(CRKP)。先前本實驗室已經找到能夠專一性感染K1 莢膜型菌株的噬菌體NTUH-K2044-K1-1 並具有全基因體定序,從中找到一為K1 莢膜分解酶的基因產物。本研究欲評估莢膜分解酶治療效果,因此接著篩選出出感染K2 莢膜型菌株的噬菌體1611E-K2-1 並從中找出K2 莢膜分解酶基因,利用基因重組技術大量表現純化出K1、K2 莢膜分解酶並證實對小鼠不具毒性,以腹腔注射感染K1、K2 莢膜型菌株的小鼠在感染後1 小時內接受單劑莢膜分解酶(25 μg)治療可提高87.5%(K1)或100%(K2)的存活率。目前將莢膜分解酶療法擴展至多重抗藥性菌株感染的治療,實驗室統計台大、長庚、成大和榮總四家醫院的CRKP 臨床菌株發現有集中於K64 莢膜型的現象。欲模擬臨床上易感染多重抗藥性菌株之免疫不全病人族群,利用Cyclophosphamide 建立免疫不全小鼠模式,以腹腔注射感染K64 莢膜型菌株後1 小時接受單劑K64 莢膜分解酶(150 μg)進行治療,仍可成功提高62.5%的存活率。因此,基因表現之莢膜分解酶應可作為傳統抗生素之外治療克雷伯氏肺炎桿菌感染的替代療法。 | zh_TW |
dc.description.abstract | Community-acquired pyogenic liver abscess (PLA) caused by Klebsiella pneumoniae has become an emerging infectious disease. The capsule of K. pneumoniae is an important virulence factor, which can keep bacteria from phagocytosis and complement mediated lysis. Previous studies reported K1 and K2 were prevalent capsular serotypes of strains causing PLA in Taiwan. Multi-drug resistances of K. pneumoniae including Extended-spectrumβ-lactam resistance and Carbapenem-resistance Klebsiella pneumoniae (CRKP) are urgent global issues. Phage NTUH-K2044-K1-1 and its capsule depolymerase which were both specific to K1 capsule were identified in our previous study. Here, we isolated a phage 1611E-K2-1 shown to infect K2 strains and identified its K2 capsule depolymerase. Intraperitoneal administration of a single dose of 25 ug K1 or K2 capsule depolymerase at 1h after intraperitoneal infection of K. pneumoniae K1 or K2 strain could enhance 87.5 %(K1) or 100 %(K2) survival rate, respectively. We further tested capsule depolymerase treatment of multi-drug resistant (MDR) K. pneumoniae infection. We observed the most prevalent capsular type of CRKP from NTUH, CGUH, NCKUH, and VGH was K64. The efficacy of K64 capsule depolymerase treatment in cyclophosphamide-treated mice mimicing the immune-compromised patients infected with MDR K. pneumoniae was evaluated. Intraperitoneal administration of a single dose of 150 ug K64 capsule depolymerase at 1h after intraperitoneal infection of K. pneumoniae K64 strain also improve 62.5 % survival. Therefore, capsule depolymerase treatment is a potential therapy for K. pneumoniae infection alternative to conventional antibiotics treatment. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T08:04:03Z (GMT). No. of bitstreams: 1 ntu-103-R01445126-1.pdf: 4878548 bytes, checksum: e90430782903810fb9f04ba3fdcfb284 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 目錄
口試委員審定書 I 致謝 II 中文摘要 III Abstract IV 第一章、緒論 1 1-1 全球性新興感染症:克雷伯氏肺炎桿菌造成之原發性肝膿瘍 1 1-2 克雷伯氏肺炎桿菌的莢膜血清分型 2 1-3 克雷伯氏肺炎桿菌的多重抗藥性菌株(multidrug resistant, MDR) 2 1-4 多重抗藥性克雷伯氏肺炎桿菌感染的抗生素治療 4 1-5 尋找新的替代療法(Alternative therapy)取代傳統抗生素治療 5 1-6 研究目的 9 第二章、材料與方法 10 2-1 實驗菌株 10 2-2 培養基 10 2-3 尋找及分離噬菌體 10 2-4 塗點試驗(Spot test) 11 2-5 噬菌體增生(Phage amplification) 11 2-6 噬菌體溶菌斑效價分析 12 2-7 噬菌體純化與DNA萃取 12 2-8 限制性核酸內切酶實驗 14 2-9 噬菌體全基因體序列分析 14 2-10 基因選殖(Cloning) 15 2-11 蛋白質純化與表現 16 2-12 SDS膠體電泳與考馬斯亮藍(coomassie brilliant blue)蛋白質定量法 18 2-13 莢膜多醣抽取法 20 2-14 莢膜多醣與莢膜分解酶作用實驗 21 2-15 實驗動物 22 2-16 以莢膜分解酶治療動物感染模式 22 2-17 西方墨點法(Western Blot) 22 2-18 血清毒殺試驗分析 23 2-19 建立免疫不全小鼠模式 24 2-20 以莢膜分解酶治療免疫不全小鼠感染模式 24 第三章、結果 25 3-1 尋找及分離可感染K2莢膜型克雷伯氏肺炎桿菌之K2噬菌體 25 3-2 1611E-K2-1溶菌斑效價分析及噬菌體增生 25 3-3 1611E-K2-1宿主範圍分析與對克雷伯氏肺炎桿菌K2莢膜型不同菌株之感染能力分析 26 3-4 獲取1611E-K2-1基因體 27 3-5 1611E-K2-1基因體定序與生物資訊分析 27 3-6 利用基因重組技術表現並純化K1-ORF33及K2-ORF16之基因產物 28 3-7 測試純化出的K1-ORF33及K2-ORF16基因產物酵素活性(enzyme activity) 29 3-8 確認K2莢膜分解酶對K2莢膜的降解切割能力 30 3-9 評估K1、K2莢膜分解酶治療對動物的安全性及副作用 30 3-10 評估K1、K2莢膜分解酶療法(capsule depolymerase treatment)的有效性 31 3-11 分析由臨床分離之抗藥性克雷伯氏肺炎桿菌菌株 31 3-12 測定克雷伯氏肺炎桿菌抗藥性菌株感染BALB/c小鼠的半致死劑量(LD50) 32 3-13 純化由噬菌體分離之K64莢膜分解酶 33 3-14 測試純化後之K64莢膜分解酶酵素活性 33 3-15 進行血清毒殺試驗分析(serum killing assay) 33 3-16 利用免疫抑制藥物(Cyclophosphamide)建立免疫不全小鼠模式並評估K64莢膜分解酶療法的有效性 34 第四章、討論 36 參考文獻 65 圖目錄 圖 一、確認K1莢膜分解酶蛋白質純化情形 42 圖 二、確認K2莢膜分解酶蛋白質純化情形 43 圖 三、K2莢膜分解酶對K2莢膜型菌株進行酵素效力試驗 44 圖 四、K2莢膜分解酶對K13莢膜型菌株進行酵素效力試驗 45 圖 五、確認K2莢膜分解酶對K2莢膜多醣的切割降解能力 46 圖 六、以西方墨點法確認注射過K1莢膜分解酶小鼠之血清抗體產生情形 47 圖 七、以西方墨點法確認注射過K2莢膜分解酶小鼠之血清抗體產生情形 48 圖 八、K1莢膜分解酶療法於NTUH-K2044(K1)克雷伯氏肺炎桿菌菌株感染BALB/c小鼠的療效評估 49 圖 九、K2莢膜分解酶療法於A4528(K2)克雷伯氏肺炎桿菌菌株感染BALB/c小鼠的療效評估 50 圖 十、分析K64莢膜型克雷伯氏肺炎桿菌ESBL臨床菌株的限制性片段長度多態性(restriction fragment length polymorphism, RFLP) 51 圖 十一、分析K64莢膜型克雷伯氏肺炎桿菌CRKP臨床菌株的限制性片段長度多態性(restriction fragment length polymorphism, RFLP) 52 圖 十二、確認K64莢膜分解酶蛋白質純化情形 53 圖 十三、K64莢膜分解酶對K64莢膜型菌株進行酵素效力試驗 54 圖 十四、血清毒殺試驗 55 圖 十五、以Cyclophosphamide藥物建立免疫不全小鼠模式期間小鼠體重變化 56 圖 十六、K64莢膜分解酶療法於7960(K64)克雷伯氏肺炎桿菌菌株感染免疫不全BALB/c小鼠的療效評估 57 表目錄 表 一、本研究使用細菌菌株及質體 58 表 二、研究中使用之引子(primer)DNA序列 59 表 三、K2噬菌體(1611E-K2-1)宿主範圍 60 表 四、National center for biotechnology information Basic Local Alignment Search Tool(NCBI BLAST)胺基酸序列資料庫,比對K2噬菌(1611E-K2-1)genome上ORFs序列之結果 61 表 五、噬菌體NTUH-K2044-K1-1之開放閱讀框K1-ORF33(K1莢膜分解酶基因序列)(1953 bp) 62 表 六、噬菌體1611E-K2-1之開放閱讀框K2-ORF16(K2莢膜分解酶基因序列)(2004 bp) 63 表 七、噬菌體K11-7-1-1之開放閱讀框S2-5(K64莢膜分解酶基因序列)(2988 bp) 64 | |
dc.language.iso | zh-TW | |
dc.title | 莢膜分解酶治療克雷伯氏肺炎桿菌感染之小鼠模式 | zh_TW |
dc.title | Utilization of capsule depolymerase as a treatment for Klebsiella Pneumoniae infection in mice | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林稚容,董馨蓮,楊宏志,蔡丰喬 | |
dc.subject.keyword | 克雷伯氏肺炎桿菌,莢膜型,噬菌體,多重抗藥性,莢膜分解?療法, | zh_TW |
dc.subject.keyword | Klebsiella pneumoniae,capsule type,phage,multi-drug resistant,capsule depolymerase treatment, | en |
dc.relation.page | 71 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2014-07-02 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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