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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張永祺 | |
| dc.contributor.author | Yi-Hsuan Chen | en |
| dc.contributor.author | 陳逸萱 | zh_TW |
| dc.date.accessioned | 2021-05-19T17:46:21Z | - |
| dc.date.available | 2023-08-01 | |
| dc.date.available | 2021-05-19T17:46:21Z | - |
| dc.date.copyright | 2018-08-01 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-07-18 | |
| dc.identifier.citation | 1 Patterson, M. J. in Medical Microbiology (eds th & S. Baron) (1996).
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7555 | - |
| dc.description.abstract | Streptococcus pyogenes又稱為A型鏈球菌 (Group A Streptococcus)，是一以人類為唯一天然宿主，可以引起咽喉炎、壞死性筋膜炎及菌血症的細菌。已知莢膜能幫助A型鏈球菌造成侵入性感染，是一重要的毒力因子。而M4血清型A型鏈球菌雖然缺少莢膜，卻是主要造成A型鏈球菌侵入性感染的血清型，顯示M4血清型A型鏈球菌可能有其他毒力因子能夠抵抗宿主的免疫系統。我們實驗室與其他研究均發現，由fibronectin-binding, collagen-binding, T antigen (FCT) region所編碼的線毛 (Pilus) 能幫助A型鏈球菌貼附到宿主的皮膚和咽喉細胞、形成生物膜並造成小鼠的皮膚感染。除此之外，實驗室之前的研究進一步發現，以腹腔注射M4血清型A型鏈球菌感染小鼠時，野生株較缺乏線毛的spy0116突變株更能引起小鼠的死亡，且野生株在人類全血中有較佳的存活率，顯示線毛的存在對M4血清型A型鏈球菌躲避免疫攻擊，並造成系統性感染有其必要性。血紅素結合蛋白 (haptoglobin) 是一種急性期蛋白，先前有研究指出可藉由與白血球表面的受器結合來調控細胞的反應。實驗室發現M4血清型A型鏈球菌的線毛能與haptoglobin結合，因此我的實驗主軸在研究 (1) 線毛本身是否能幫助M4血清型A型鏈球菌抵抗宿主的免疫系統，(2) M4血清型A型鏈球菌是否可以藉由鍵結haptoglobin來抑制宿主抗菌反應。實驗發現野生株在血清中有存活較好的趨勢，但是野生株及突變株被補體標記的程度相當且野生株對於抗菌胜肽較敏感，顯示線毛本身不足以使野生株較利於存活。然而預先與haptoglobin結合的野生株較能抵抗血清、血小板和嗜中性白血球胞外網狀結構 (Neutrophil extracellular traps，NETs) 的殺菌作用，進而使M4血清型A型鏈球菌野生株在全血中存活較好。Siglec-9是一種能和唾液酸結合的抑制受器 (inhibitory receptor)，主要表現在人類巨噬細胞和嗜中性白血球表面。我們先前發現Siglec-9可以和其鏈具有大量的唾液酸修飾的haptoglobin結合，因此我們推測M4血清型A型鏈球菌可能利用haptoglobin去與Siglec-9鍵結並抑制免疫反應。為了驗證我們的假說，我們建立了能夠穩定表現人類Siglec-9的RAW/Sig9細胞株及及控制組RAW/vector，之後將同時使用人類嗜中性白血球為實驗模型來驗證我們的假說，確認M4血清型A型鏈球菌與haptoglobin結合後是否能透過與Siglec-9結合來增加存活率。雖然尚無法確認M4血清型A型鏈球菌是否可利用haptoglobin與免疫細胞上的Siglec-9結合來幫助存活，但是我們的實驗結果仍顯示M4血清型A型鏈球菌利用線毛與haptoglobin結合是M4血清型A型鏈球菌能造成侵入性疾病的重要機轉。 | zh_TW |
| dc.description.abstract | Streptococcus pyogenes (group A streptococcus, GAS) is a human pathogen causing various diseases such as pharyngitis, necrotizing fasciitis and bacteremia. It is known that hyaluronic acid capsule (HA capsule) of GAS is an important virulence factor contributing to its invasive infection. GAS serotype M4 (M4 GAS), lacking the important HA capsule, is one of the major serotype causing invasive GAS infection. These observations indicate that M4 GAS may have alternative virulence factors to counteract host defense mechanisms. We and others found that pilus encoded by the fibronectin-binding, collagen-binding, T antigen (FCT) region is dedicated in host skin and pharynx adherence, biofilm formation and mice skin infection. Our previous data further demonstrated that M4 GAS wild-type (WT) causes more mortality in mice intraperitoneal infection and survives better in human blood compare to M4 GAS spy0116 mutant deficient in pilus formation. These data suggest that the pilus of M4 GAS contributes to host immune evasion and systemic infection. Haptoglobin is an acute phase protein that have been reported to regulate leukocyte functions. Our previous data showed that M4 GAS binds haptoglobin via its protruding pilus; therefore, my specific aims of this project are to investigate whether (1) M4 GAS pilus its self can counteract host immune system, (2) haptoglobin bound M4 GAS can downregulate host antibacterial responses. Our data showed that M4 GAS WT survives better in human serum than the M4 GAS spy0116 mutant. However, comparable complement deposition between M4 GAS WT and spy0116 mutant and increased AMP sensitivity in M4 GAS WT was observed, which suggests that pilus itself is not sufficient to confer to the whole survival advantage of M4 GAS WT. When precoated with haptoglobin, M4 GAS WT was more resistant to serum antimicrobials, plalets and NET-mediated killing, which together resulted in the better survival in the human whole blood. We previously found that Siglec-9, a sialic acid-binding inhibitory receptor expressed primarily on human macrophages and neutrophils, can bind to the heavily siaylatedchain of haptoglobin, which lead to the hypothesis that M4 GAS may suppress immune activation via engaging Siglec-9 with pilus bound haptoglobin. To address this question, we generated Siglec-9-expressing RAW264.7 stable cell line, and will apply this system together with human primary neutrophil system to directly address the role of Siglec-9 and haptoglobin in the immune suppression upon GAS infection. Although the detailed mechanism has not been fully elucidated, our results clearly demonstrated the binding of haptoglobin to M4 GAS pilus is essential to provide the beneficial survival advantage to subvert host antibicrobial immune responses. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:46:21Z (GMT). No. of bitstreams: 1 ntu-107-R05445126-1.pdf: 3285826 bytes, checksum: 749a28ed7f95f83f159fb96531b3ce29 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 口試委員審定書 i
致謝 ii 中文摘要 iii Abstract v 目錄 vii 第一章、 研究背景與動機 1 一、 A型鏈球菌 1 1. A型鏈球菌 (Group A Streptococcus，GAS) 1 2. A型鏈球菌之流行病學 1 3. A型鏈球菌之毒力因子 2 4. A型鏈球菌之線毛 (pilus) 3 5. A型鏈球菌之莢膜 4 二、急性期蛋白 (Acute phase proteins，Apps) 5 1. 急性期蛋白 5 2. 血紅素結合蛋白 (haptoglobin，Hp) 5 三、 Sialic acid-binding immunoglobulin-like lectins (Siglecs) 受體 6 1. Siglec受體 6 2. Siglec-9 7 四、 研究動機 8 第二章、 研究材料與實驗方法 10 一、 研究材料 10 1. 實驗菌株 (Bacteria strain) 10 2. 細胞株 (Cell lines) 11 3. 引子 (Primer) 11 4. 質體 (Plasmid) 12 5. 抗體 (Antibody) 13 6. 抗菌胜肽 (Antimicrobial peptide) 14 7. 酵素免疫分析法試劑組 14 8. 萃取細菌細胞壁之試劑 14 9. 銀染 (Silver stain) 之試劑 14 二、 實驗方法 15 1. 核酸萃取 15 2. DNA洋菜膠電泳 (DNA agarose electrophoresis) 16 3. 聚合酶連鎖反應 (Polymerase Chain Reaction，PCR) 16 4. 勝任細胞的製備及電穿孔作用 17 5. 同源置換 (Homologous recombination) 18 6. 預先結合GAS與haptoglobin 18 7. 預先使用唾液酸酶處理GBS 18 8. GAS與血清中之haptoglobin結合試驗 19 9. 流式細胞儀分析與流式細胞分選 19 10. 生物膜 (biofilm)生成試驗 21 11. 全血殺菌試驗 22 12. 血清與血漿殺菌試驗 22 13. 血小板殺菌試驗 23 14. 抗菌胜肽抑菌試驗 23 15. 組織蛋白抑菌試驗 23 16. 嗜中性白血球殺菌試驗 24 17. NETs殺菌試驗 24 18. 細胞感染之基因表現量測定 25 19. RAW細胞之細胞激素表現量測定 26 20. RAW細胞之殺菌試驗 26 21. 細胞壁萃取物之分析 26 22. 小鼠皮下感染 27 23. RAW/vector、RAW/Sig9、RAW/R120K細胞株的建立 28 第三章、 研究結果 30 一、 建立M4血清型A型鏈球菌Dspy0116突變株之互補菌株和臨床分離M4血清型A型鏈球菌之Dspy0116突變株 30 1. 建立M4血清型A型鏈球菌Dspy0116突變株之互補菌株並確認其表現 30 2. 建立剔除spy0116的臨床分離菌株並確認其表現型 31 二、 線毛在M4血清型A型鏈球菌抵抗免疫系統之角色 32 1. 具線毛之M4血清型A型鏈球菌野生株在人類全血中存活較佳 32 2. 線毛不會增加M4血清型A型鏈球菌對補體、抗菌胜肽和組織蛋白的抗性 32 3. 線毛不影響M4血清型A型鏈球菌與NETs反應後之存活率 34 4. 線毛不影響M4血清型A型鏈球菌與血小板反應後之存活率沒有差別 34 5. M4血清型A型鏈球菌Dspy0116突變株之互補菌株和臨床分離之M4血清型A型鏈球菌野生株在人類全血中之存活率 34 6. M4血清型A型鏈球菌Dspy0116突變株之互補菌株及控制組之LL-37結合能力沒有差別 35 三、 M4血清型A型鏈球菌利用線毛與血紅素結合蛋白結合來抵抗免疫系統攻擊 35 1. M4血清型A型鏈球菌的線毛能與人類血清中之haptoglobin結合 35 2. 預先與haptoglobin結合能幫助M4血清型A型鏈球菌在人類全血和血清中存活 36 3. Haptoglobin無法幫助M4血清型A型鏈球菌抵禦C3補體標記和抗菌胜肽 36 4. 預先與haptoglobin結合能幫助M4血清型A型鏈球菌抵抗NETs和血小板的殺菌作用 37 四、 M4血清型A型鏈球菌與血紅素結合蛋白結合後，可能藉由與Siglec-9鍵結來調控免疫反應 37 1. 建立RAW/vector和RAW/Sig9細胞株 37 2. 與haptoglobin結合可增加M4血清型A型鏈球菌之存活率，但RAW細胞是否表現Siglec-9不影響M4血清型A型鏈球菌之存活 39 第四章、 討論與未來研究方向 40 參考文獻 44 圖表目錄 50 附錄 68 | |
| dc.language.iso | zh-TW | |
| dc.title | 探討無莢膜之M4血清型A型鏈球菌躲避免疫系統的機制 | zh_TW |
| dc.title | Characterization of the Immune Evasion Mechanisms of the Nonencapsulated M4 Group A Streptococcus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 朱清良,顧家綺 | |
| dc.subject.keyword | A型鏈球菌,線毛,侵入性感染,血紅素結合蛋白,Siglecs, | zh_TW |
| dc.subject.keyword | Group A Streptococcus,pilus,invasive infection,haptoglobin,Siglecs, | en |
| dc.relation.page | 71 | |
| dc.identifier.doi | 10.6342/NTU201801671 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2018-07-19 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-08-01 | - |
| 顯示於系所單位： | 微生物學科所 | |
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