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系統識別號 U0026-2101201109221800
論文名稱(中文) 創傷弧菌RTX毒素在該菌之致病機制中所扮演的角色
論文名稱(英文) The role of RTX toxin in Vibrio vulnificus pathogenesis
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 99
學期 1
出版年 100
研究生(中文) 羅宏仁
研究生(英文) Horng-Ren Lo
學號 s5890107
學位類別 博士
語文別 中文
論文頁數 79頁
口試委員 召集委員-林以行
口試委員-吳俊忠
口試委員-鄧景浩
口試委員-王錦堂
口試委員-林銘德
指導教授-何漣漪
中文關鍵字 創傷弧菌  RTX毒素  細胞毒性  移生  吞噬作用 
英文關鍵字 Vibrio vulnificus  RTX toxin  cytotoxicity  colonization  phagocytosis 
學科別分類
中文摘要 創傷弧菌 (Vibrio vulnificus) 是一種嗜鹽性革蘭氏陰性細菌,可引起傷口感染以及菌血症。創傷弧菌RTX毒素對各種類型的細胞株都具有毒殺性,是細菌重要的毒素。我們建構一個RTX基因刪除之突變株 (rtxA1),以此來探討此細胞毒素在創傷弧菌致病過程所扮演的角色,結果顯示在小鼠感染模式下,無論是以何種注射感染的方式,此突變株的毒力都減少約100倍。小鼠以皮下注射的方式感染了野生株後,可以觀察到其肌肉、皮下脂肪以及真皮的結締組織皆呈現嚴重的破壞;感染rtxA1突變株的小鼠雖然其感染部位有吞噬細胞浸潤,但並沒有組織破壞的現象。由小鼠背部氣囊感染細菌的實驗結果顯示,rtxA1突變株在感染處移生 (colonization) 的能力較野生株來得差,因此在血液和體內臟器的細菌數均低於野生株,其逆轉株 (revertant) 則恢復與野生株類似的感染能力。若以cyclophosphamide處置小鼠使嗜中性白血球數目低落,則可提高rtxA1突變株對小鼠的毒力以及在感染處的存活,對於感染部位的組織傷害也近似於野生株,並提高突變株在血液以及在老鼠體內臟器的存活。我們進一步發現,不產生RTX毒素的創傷弧菌突變株,無論是在吞噬細胞浸潤的小鼠腹腔中或是與吞噬細胞株RAW264.7共同培養,其存活數都比具有正常功能的RTX菌株低。由此我們推論,創傷弧菌之RTX促進該菌移生的作用,可能與其減弱吞噬細胞清除細菌的能力有關。
英文摘要 Vibrio vulnificus is a halophilic gram-negative bacterium causing wound infection and bacteremia in human. An RTX toxin produced by this pathogen can lyse a variety of cells and is required for bacterial virulence. To investigate the role of RTX in V. vulnificus pathogenesis, an RTX-deficient mutant, rtxA1, was constructed. This mutant showed about 100-fold reduced virulence in mice challenged via various routes. The mouse infected subcutaneously by the wild type strain showed severely damaged adipose, muscle and connective tissue; however, infection by the rtxA1 mutant resulted in phagocytes infiltration but no obvious tissue destruction. In an air-sac infection model, the rtxA1 mutant was much less able to colonize at the infection site and invade into the bloodstream and internal organs than the parent and revertant strains. The colonization and virulence ofrtxA1 mutant in the mouse were improved when the neutrophils were depleted by cyclophosphamide treatment. The ability of rtxA1 mutant to cause tissue damage and spread into bloodstream and internal organs were also comparable to that of the parent strain in the neutropenic mouse. Furthermore, the RTX-deficient mutants survived much poorly in the phagocyte-enriched peritoneum and were more readily internalized by the mouse macrophage cell line, RAW264.7 than the RTX-proficient strains. These suggest that the RTX toxin could promote bacterial colonization probably by impairing the phagocytic ability of the phagocytes.
論文目次 中文摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vii
致謝 x
緒論 1
創傷弧菌的生物特性 1
創傷弧菌感染症的病徵 1
創傷弧菌的分型 2
創傷弧菌的毒力因子 4
莢膜多醣體 5
附著因子 6
鐵利用能力 6
細胞溶解素 (vvhA) 和金屬蛋白酶 (vvp) 7
RTX毒素 9
先天性免疫在創傷弧菌感染所扮演的角色 13
實驗目的 14
材料與方法 15
1. 實驗菌株、質體及實驗動物 15
1-1. 實驗菌株 15
1-2. 質體 15
1-3. 寡聚核苷酸引子 15
1-4. 細胞培養與老鼠週邊血嗜中性白血球分離 16
1-5. 實驗動物 16
2. DNA 萃取與操作 17
2-1. Genomic DNA 萃取 17
2-2. 質體 DNA 萃取 17
2-3. 限制酶反應 18
2-4. DNA 去磷酸化作用 19
2-5. DNA 接合反應 19
3. 細胞轉型試驗 20
3-1. 熱休克轉形作用所需勝任細胞製備 20
3-2. 熱休克細胞轉型反應 20
3-3. 電穿孔轉型作用(Electroporation)所需勝任細胞製備 20
3-4. 電穿轉型作用 21
4. 接合生殖作用 21
5. 聚合酶連鎖反應(polymerase chain reaction; PCR) 22
6. 南方墨點法(Southern hybridization) 22
7. DNA 定序與分析 23
8. 創傷弧菌突變株與逆轉株(revertant)之製備 23
9. 細菌生長曲線之測定 24
10. 細菌對人類血清殺菌作用之抗性 25
11. 金屬蛋白酶、細胞溶解毒素活性之測試 25
11-1金屬蛋白酶活性測試 25
11-2. 細胞溶解毒素活性測試 26
12. 細胞毒殺性分析 26
13. 毒力試驗 27
14. 細菌侵襲力的評估 27
15. 組織學分析 27
16. 細菌移生能力分析(colonization assay) 28
17. 活體感染影像系統 (In vivo image analysis; IVIS) 28
18. 吞噬試驗(phagocytosis assay) 29
結果 30
rtxA1 突變株在細胞毒殺性與對小鼠的毒力 30
rtxA1 突變株對小鼠組織的傷害 31
rtxA1 突變株之移生能力及侵襲力 32
rtxA1突變株在嗜中性白血球低落的小鼠之組織傷害、移生能力和毒力 33
rtxA1 與抗吞噬作用之相關性 35
討論 37
未來展望 45
參考文獻 64
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