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系統識別號 U0026-2608201303320900
論文名稱(中文) 探討困難梭狀桿菌所引起之炎症小體活化現象
論文名稱(英文) Study on Clostridium difficile-induced inflammasome activation
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 101
學期 2
出版年 102
研究生(中文) 張詠琪
研究生(英文) Yung-Chi Chang
學號 T36004071
學位類別 碩士
語文別 英文
論文頁數 50頁
口試委員 召集委員-賴信志
指導教授-蔡佩珍
口試委員-黃一修
中文關鍵字 困難梭狀桿菌  炎症小體  P2X7受體  MyD88  pyroptosis 
英文關鍵字 C. difficile  inflammasome  P2X7 receptor  MyD88  pyroptosis 
學科別分類
中文摘要 困難梭狀桿菌感染是目前主要導致院內感染的重要致病菌之一,住院病人因長期服用抗生素易導致其感染而引發感染性腹瀉、偽膜性大腸炎等症狀。目前認為其致病機轉主要是因宿主體內產生過度的炎症反應所導致,過去的報導中也曾指出病人體內可測得相當大量的前發炎性細胞激素,例如IL-1β等。目前已知炎症小體(inflammasome)所調控活化的Caspase-1可切割出具有生理活性功能的IL-1β,此外也會導致一種特殊的細胞凋亡形式,稱之為pyroptosis。而根據目前的文獻顯示,關於困難梭狀桿菌與炎症小體之間的調控機制仍有許多爭議跟未知的地方存在,故本研究主要想要探討炎症小體在困難梭狀桿菌感染當中所扮演的角色及其詳細的調控機制。首先,我們發現細菌感染腹腔巨噬細胞後可藉由Caspase-1活化而引發大量IL-1β的產生,並隨感染的劑量及時間增加而越趨明顯。此外,具毒素的菌株在細胞內可藉由調控P2X7受體引發炎症小體活化以及Caspase-1誘發之pyroptosis現象發生,進一步導致細胞膜破損及胞內物質釋放,例如LDH、HMGB1等。特別的是,我們也發現細菌會從pyroptotic細胞被釋放。而在MyD88-/-、Tlr2-/-和Tlr2-/-/Tlr4-/-的細胞中,pro-IL-1β的基因表現量以其IL-1β的產生都有明顯下降,但在Tlr4-/-的細胞中則無明顯差異。為了證實其在宿主當中的機轉,我們看到小鼠在感染後腸道內也有炎症小體活化現象,並且當其受抑制之後會導致腸道發炎減少以及菌量變多。綜合上述,我們的研究結果顯示,困難梭狀桿菌感染可經由MyD88所調控的TLR2訊息路徑誘導pro-IL-1β的產生,並且毒素菌株可在細胞內進一步引發P2X7受體依賴性之炎症小體活化以及pyroptosis現象產生,而此現象對於宿主在面對困難梭狀桿菌感染當中可能扮演重要的免疫防禦機制。
英文摘要 Clostridium difficile infection (CDI) is the major leading cause of nosocomial infection in hospitalized patients with long-term antibiotic treatment. An excessive host inflammatory response is believed to be the major mechanism in pathogenesis of C. difficile infection and various proinflammatory cytokines such as IL-1β are detected in patients with C. difficile infection. IL-1β is known to be processed by caspase-1, a cysteine protease regulated by a protein complex called inflammasome, which leads to a specialized form of cell death, pyroptosis. However, the precise mechanism of C. difficile induced- inflammasome activation is still controversial and unclear. Thus, the specific aim of this study is to assess the role of inflammasome and its detail mechanism during C. difficile infection. First, we found that caspase-1 dependent IL-1β production was induced by C. diffiicle in peritoneal macrophages, and increased in a dose- and time-dependently manner under aerobic condition. Moreover, intracellular toxigenic C. diffiicle is essential to P2X7–dependent inflammasome activation and subsequent caspase-1-dependent pyroptotic cell death leading to loss of membrane integrity and release of intracellular contents, such as LDH and HMGB1. Notably, we also observed bacteria were released from pyroptotic cells. In addition, the mRNA expression of pro-IL-1β and IL-1β production was significantly reduced in MyD88-/-, Tlr2-/- and Tlr2-/-/Tlr4-/- cells, whereas comparable in Tlr4-/- cells compared with wild-type cells. Finally, to investigate the role of inflammasome in vivo, we found that colonic inflammasome activation was also induced by CDI, and caspase-1 inhibition led to less intestinal inflammation and increased load of C. difficile. Taken together, our study suggested that MyD88-mediated TLR2 signaling was involved in pro-IL-1β production and intracellular toxigenic C. diffiicle induced following P2X7- dependent inflammasome activation and pyroptosis which may act as an important regulation in host defence during C. difficile infection.
論文目次 中文摘要....................................................I
ABSTRACT..................................................II
致謝.....................................................III
CONTENTS..................................................IV
INDEX OF TABLES AND FIGURES...............................VI
INTRODUCTION...............................................1
Epidemiology of Clostridium difficile infection............1
Pathogenesis of Clostridium difficile infection............2
Host immune responses to Clostridium difficile infection...3
Importance of inflammasome in bacterial recognition........5
Mechanism of inflammasome activation.......................6
Significance of host cell death during infection...........7
The role of pyroptosis during bacterial infection..........8
MATERIALS AND METHODS......................................9
Bacteria strain............................................9
Animal.....................................................9
Bacterial culture..........................................9
Extraction of surface layer proteins (SLPs)...............10
Reagent and cell line.....................................10
Isolation of peritoneal macrophages.......................11
In-vitro cell infection...................................11
Assessment of IL-1β production by ELISA...................12
Cytotoxicity assay (LDH assay)............................12
Preparation of cell culture supernatant and cell protein lysates...................................................12
Tissue protein extraction.................................13
Western blotting..........................................13
Total RNA extraction......................................14
Reverse transcription and real-time PCR...................15
C. difficile infection animal model.......................15
In vivo imaging...........................................16
Bacterial burden..........................................16
Data analysis.............................................17
RESULTS...................................................18
Mature IL-1β and caspase-1 was induced by C. difficile infection.................................................18
C. difficile induced caspase-1 dependent IL-1β production.18
C. difficile induced P2X7-dependent inflammasome activation................................................19
Intracellular bacterial toxin is essential to inflammasome activation during CDI.....................................20
Hypertoxigenic C. difficile induced increased inflammasome activation and more severe macrophages damage.............21
Toxigenic C. difficile induced caspase-1-dependent pyroptotic cell death.....................................22
MyD88-mediated TLR2 signaling pathway was involved in C. difficile induced pro-IL-1β production....................24
Colonic inflammasome was activated by C. difficile infection.................................................25
The role of caspase-1-dependent inflammasome activation in host defense during CDI...................................25
DISCUSSION................................................27
REFERENCES................................................32
APPENDIX..................................................48
REAGENTS LIST............................................50
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