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| 研究生： |
邱柏蓉 Chiu, Po-Jung |
|---|---|
| 論文名稱： |
探討困難梭狀芽孢桿菌孢子與巨噬細胞的交互作用 Insights into Clostridium difficile spore and macrophage interaction |
| 指導教授： |
黃一修
Huang, I-Hsiu |
| 學位類別： |
碩士 Master |
| 系所名稱： |
醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology |
| 論文出版年： | 2019 |
| 畢業學年度： | 107 |
| 語文別： | 英文 |
| 論文頁數： | 58 |
| 中文關鍵詞： | 困難梭狀芽孢桿菌 、孢子 、巨噬細胞 |
| 外文關鍵詞： | Clostridium difficile, spore, macrophage |
| 相關次數： | 點閱：47 下載：0 |
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困難梭狀芽孢桿菌是革蘭氏陽性，會形成孢子的絕對厭氧細菌，並且是全世界抗生素相關性腹瀉（AAD）的主要原因。在困難梭狀芽孢桿菌感染（CDI）期間，休眠中的孢子將萌發、定殖在結腸中並產生毒素。受到困難梭狀芽孢桿菌感染的過程中，會有大量的巨噬細胞和嗜中性顆粒細胞的聚集，這表明困難梭狀芽孢桿菌與先天免疫系統之間必然存在某種交互作用。早期對於困難梭狀芽孢桿菌感染的研究主要集中在探討其營養體細胞和巨噬細胞之間的相互作用上。我們假設外來的孢子也可能會與巨噬細胞相互作用。在這項研究中，我們確認了困難梭狀芽孢桿菌和RAW 264.7巨噬細胞之間的相互作用。首先，我們使用吞噬作用試驗和透射式電子顯微鏡確定RAW 264.7細胞確實能夠攝取來自產毒分離株，菌株R20291，DPS630，VPI10463和非產毒分離株，菌株37780，TNHP1和TNHP3的困難梭狀芽孢桿菌孢子。根據孢子存活試驗顯示，孢子可以存活在細胞內，並且在共培養48小時後，孢子仍具有活性。除此之外，我們也觀察了孢子對細胞的毒殺姓，結果顯示經過24小時共培養後，產毒與非產毒株的孢子對巨噬細胞均具有毒性。更進一步，我們研究了在困難梭狀芽孢桿菌孢子感染期間RAW 264.7細胞被誘導產生的炎性細胞因子。使用促炎細胞因子的酵素結合免疫吸附分析法和定量即時聚合酶鏈鎖反應測定，我們觀察到與來自非產毒菌株的孢子相比，產毒菌株的孢子能夠誘導更高的炎性細胞因子產生。使用同基因毒素突變體，我們證明炎性細胞因子的誘導需要存在毒素A或B編碼基因。總而言之，產毒菌株孢子的存在可在小鼠巨噬細胞RAW 264.7細胞中誘導強烈的促炎細胞因子反應，並歸因於孢子本身攜帶毒素。
Clostridium difficile (C. difficile) is a Gram-positive, spore-forming anaerobic bacterium and is the leading cause of antibiotic associated diarrhea (AAD) worldwide. During C. difficile infections (CDI), dormant spores in the colon will germinate, colonize and produce toxins. The massive macrophage and neutrophil recruitment during the course of CDI suggests that there must be some sort of interaction between C. difficile and the innate immune system. Studies on the early events of C. difficile infections have been largely focused on the interactions between vegetative cells and macrophages. We hypothesized that spores might also interact with macrophages. In this study, we examined the interactions between C. difficile spores and RAW 264.7 macrophages. First, using phagocytosis assay and transmission electron microscopy, we determined that RAW 264.7 cells were able to interact and ingest C. difficile spores from toxigenic isolates, strains R20291, DPS630, VPI10463 and non-toxigenic isolates, strains 37780, TNHP1 and TNHP3. The survival assay showed that intracellular spores remain viable even after 48-hour post co-incubation. In addition, we also evaluate the cytotoxicity of spores in RAW 264.7 cells, we determine that spores from toxigenic and non-toxigenic are cytotoxic to macrophages after 24-hours co-incubation. Further, we investigate the induction of inflammatory cytokines by RAW 264.7 cells during the infection of C. difficile spores. Using pro-inflammatory cytokine ELISA assay and real-time qPCR, we observed that spores from toxigenic strains were able to induce higher inflammatory cytokines production compared to spores from non-toxigenic strains. Using isogenic toxin mutants, we demonstrated that the induction of inflammatory cytokines required the presence of either toxin A or B encoding genes. We hypothesize that during spore assembly, cytosolic toxins might be incorporated onto the surface of spores in trace amounts, and these toxins are sufficient to induce inflammatory response. In summary, the presence of toxigenic strain spores can induce robust pro-inflammatory cytokine response in murine macrophage RAW 264.7 cells and is attributed to the carrying of toxins by spore itself.
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