臺灣博碩士論文加值系統

創傷弧菌是一種侵入性細菌病原，透過傷口感染或食入汙染海鮮，可造成嚴重的皮膚損傷與敗血症。本實驗室發現該菌所產生的一種稱為RTX的細胞毒素可能藉由對抗吞噬細胞而促進創傷弧菌在感染部位的增殖，進而侵入血流。本論文研究進一步探討RTX毒素如何達成對抗吞噬細胞的功用。我們比較創傷弧菌野生株YJ016與其不產RTX的突變株HL128在殺死吞噬細胞、被吞噬細胞吞噬的數量、在吞噬細胞中的存活力等方面的差異。結果發現，YJ016在moi = 1的條件下，與小鼠巨噬細胞株RAW 264.7細胞共同培養 90分鐘時仍維持細胞膜的完整性。在此狀況下，YJ016的總存活菌數比HL128高約4倍。而以gentamicin保護試驗或?啶橙-結晶紫染色鏡檢法評估被吞入RAW 264.7細胞內的菌數，則YJ016比HL128低約4倍。不過，YJ016與HL128被吞噬後，在RAW 264.7細胞內的存活率皆相似。使用低溫4℃或cytochalasin D抑制吞噬細胞吞噬作用，可以看到HL128在細胞內菌量因此減少，並且以cytochalasin D抑制吞噬細胞的吞噬能力後，可觀察到HL128的總存活數與YJ016相似。因此，RTX可以保護創傷弧菌對抗巨噬細胞的吞噬作用，但對已被吞噬的細菌，則無提高其胞內存活力的作用。以創傷弧菌感染由小鼠腹腔回收的巨噬細胞，再以?啶橙-結晶紫染色觀察細菌被吞噬的數量，得到與感染RAW 264.7細胞株時同樣的結果。另一方面，無莢膜突變株JF046感染RAW 264.7細胞後，總存活數與HL128相當，但胞內菌量則明顯較HL128為低。當我們進一步將JF046的rtxA基因刪除後所得到的不產莢膜與RTX的雙突變株YH001在與RAW 264.7共同培養時，其總存活數顯著下降且胞內菌量甚至較HL128為高，顯示在無血清補體作用的條件下，RTX對創傷弧菌抗吞噬細胞吞噬的能力比莢膜重要。

Vibrio vulnificus, an invasive bacterial pathogen, causes severe skin lesions and septicemia in humans via wound infection or ingestion of contaminated seafood. Previous studies in this laboratory have shown that the RTX toxin of V. vulnificus promotes bacterial colonization at the infection site and subsequent invasion into the bloodstream by countering the infiltrating phagocytes. In this study we compared the total surviving and phagocytosed bacterial numbers, and the intracellular survival rate in phagocytes between the wild type strain, YJ016, and ΔrtxA mutant, HL128, to understand how RTX contributes to antiphagocytosis. We found that when coincubated with the mouse macrophage cell line RAW 264.7 at a moi of 1 for 90 min, conditions in which the cells remained viable, YJ016 survived about four-fold better than HL128. On the other hand, compared to YJ016, nearly four-fold more of HL128 were ingested into the RAW 264.7 cells in the gentamicin protection assay, which is consistent with the results of acridine orange-crystal violet stain for detecting the intracellular bacteria. Nevertheless, the phagocytosed bacteria of both YJ016 and HL128 were killed at similar rates. Inhibition of phagocytosis of RAW 264.7 cells by low temperature or cytochalasin D treatment resulted in reduced intracellular number of HL128, and the total survival of HL128 increased to the wild-type level after cytochalasin D treatment of the phagocytes. These results suggest that RTX may prevent phagocytosis of V. vulnificus by the macrophages but have little contribution to the survival of phagocytosed bacteria. The phagocytosis of various V. vulnificus strains by the peritoneal macrophages was similar to that by the RAW264.7 cells when examined by acridine orange-crystal violet stain. The acapsular mutant, JF046, and HL128 survived equally well in the presence of RAW 264.7 cells, but much lower number of JF046 was phagocytosed. We further deleted rtxA from JF046 to obtain an acapsular, ΔrtxA mutant, YH001. The survival of this double mutant in the presence of RAW 264.7 was significantly reduced, and the phagocytosed bacterial number of this mutant was even higher than that of HL128. These results suggest that RTX is more important than the capsule in protecting the unopsonized V. vulnificus from phagocytosis.

中文摘要 I
Abstract II
誌謝 III
目錄 IV
表目錄 VI
圖目錄 VII
符號及縮寫 VIII
緒論 1
材料與方法 6
I、實驗菌株、質體及其保存方法 6
II、實驗細胞株培養與保存 6
III、實驗方法 7
1、細胞培養與製備方法 7
(1) 細胞株 (cell line) 7
(2) 小鼠腹腔沖洗細胞 (mouse peritoneal exudates；PECs) 7
2、細胞毒性實驗 (Cytotoxicity test) 8
3、吞噬細胞吞噬實驗 (Phagocytosis killing assay) 8
(1) 細菌總存活數 9
(2) 吞噬細胞吞噬菌量 9
(3) 被吞噬細菌存活數 9
(4) 吞噬抑制實驗 9
4、?啶橙-結晶紫染色 (Acridine orange-crystal violet stain) 10
5、突變株之建構與分離 10
(1) 接合作用 (Conjugation) 10
(2) 蔗糖培養基與抗生素培養基篩選 11
(3) 聚合?連鎖反應 (polymerase chain reaction; PCR) 11
(4) 洋菜膠電泳分析 (Agarose gel electrophoresis) 12
(5) 菌落型態 (characteristic of bacteria) 12
6、生長曲線 (Growth curve) 12
7、細菌黏附細胞之能力 (Bacteria adhesion assay) 12
結果 13
I. RTX對吞噬細胞的毒殺性 13
(1) RTX與cytolysin突變株對不同吞噬細胞之毒殺性 13
(2) RTX與cytolysin突變株在不同moi下對RAW 264.7細胞之毒殺性 13
II. RTX對創傷弧菌抗吞噬作用的貢獻 14
(1) 野生株與ΔrtxA突變株菌感染RAW 264.7細胞時細菌的存活 14
(2) 野生株與ΔrtxA突變株感染RAW 264.7細胞時被吞噬的菌量 14
(3) 野生株與ΔrtxA突變株被吞噬後在RAW 264.7細胞內的存活能力 15
(4) 野生株與ΔrtxA突變株感染小鼠腹腔巨噬細胞吞噬作用的情形 15
(5) 生物二型野生株與ΔrtxA突變株感染RAW 264.7細胞時被吞噬的菌量 15
(6) 吞噬抑制實驗 16
III. RTX與莢膜在創傷弧菌抗吞噬作用上功能的差異 16
(1) 突變株的確認，生長曲線與對細胞的黏附能力 17
(2) RTX與莢膜對創傷弧菌存活與被吞噬菌量的影響 17
討論 18
參考文獻 23
圖表集 27
附錄 48
自述 50

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