許秉寧Hsu, Ping-Ning臺灣大學:免疫學研究所陳姵璇Chen, Pei-HsuanPei-HsuanChen2010-05-102018-07-092010-05-102018-07-092008U0001-2807200816260800http://ntur.lib.ntu.edu.tw//handle/246246/181794The Gram-negative bacterium Helicobacter pylori causes gastritis, gastric ulceration, and gastric cancer in humans. The key pathophysiological event in H. pylori infection is initiation of an inflammatory response, including polymorphonuclear and mononuclear cell infiltration, such as macrophages, and proinflammatory cytokine production in gastric mucosa. One of the most important mediators of inflammation is Interleukin (IL)-1β. It is reported that the inflammasome, which comprises nucleotide and oligomerization domain-like receptor (NLR), apoptosis-associated speck-like protein containing a CARD (ASC) and procaspase-1, is a molecular platform which triggers caspase-1 activation and pro-IL-1β processing. Moreover, recent studies demonstrate that certain pathogens such as Salmonella typhimurium, Listeria monocytogenes and Staphylococus aureus are potent activators of the inflammasome. However, whether the inflammasome could be activated in response to H. pylori is still unknown. In order to examine whether H. pylori could induce IL-1β production, we first detected IL-1β in the culture medium of H. pylori-cocultured THP-1 cells or murine macrophages by IL-1β ELISA. To further confirm H. pylori-induced IL-1β release was through caspase-1, we detected the activation of caspase-1 by Western blot. Our results demonstrated that H. pylori could induce caspase-1 activation and IL-1β release, indicating H. pylori could activate the inflammaseome. In addition, different H. pylori strains revealed different ability to IL-1β production and these clinical isolates could induce IL-1β production. The clinical strains isolated from gastric ulcer are with lower IL-1β inducing activity and the clinical isolated strain from gastric cancer is with the higher IL-1β inducing activity compared to other strains. Since H. pylori is a potent activator of the inflammasome, the question of which constituents sensed by the inflammasome arises. Thus, we investigated which component of H. pylori is involved in the activation of inflammasome by screening H. pylori mutants. Most of H. pylori strains contain the virulence factor, Cag (cytotoxin-associated genes) A. Our results revealed that the production of IL-1β is reduced via mutants with cagA deletion, indicating H. pylori CagA protein involves in inflammasome activation and IL-1β production. Activation of inflammasome may play a role in inflammatory response induced by H. pylori, and the virulent factor CagA is critical in H. pylori-induced inflammasome activation.Table of Contentscknowledgement ibstract(Chinese) iibstract ivable of Contents vihapter Ⅰ Introduction 1art 1. Helicobacter pylori and its pathogenesis 1art 2. Inflammasomes activation in inflammation 3art 3. Infection and Inflammasomes activation 5art 4. Aim of the study： Investigate whether H. pylori could induce IL-1β production through the activation of inflammasome. 7hapter Ⅱ Material and Methods 9art 1. Experimental materials 9.1 Cell line and bacteria 9.2 Bone-marrow-derived macrophages (BMDM) 10.3 Antibodies 10.4 Medium and buffer 11.5 Chemicals 12.6 General materials 15.7 Instruments 17art 2. Experimental procedures 18.1 Cytokine ELISAs 18.2 Generation of cagA gene knock out H. pylori mutant strain 18.3 Isolation of cell extracts 19.4 Western blot 19.5 Statistical analysis 20hapter Ⅲ Results 21art 1. H. pylori induces IL-1β production and caspase-1 activation. 21art 2. To characterize the induction of IL-1β production in different clinical isolated H. pylori strains. 22art 3. Role of H. pylori CagA in IL-1β production. 23hapterⅣ Discussion 25art 1. H. pylori triggers inflammasome activation. 25art 2. Role of H. pylori CagA in inflammasome activation. 27art 3. The correlation between IL-1β production and H. pylori-associated diseases. 28art 4. Conclusion 30eference 32igures 39igure 1 H. pylori induces IL-1β production in THP-1 cells. 39igure 2 H. pylori induces IL-1β production in bone marrow derived macrophages (BMDMs). 40igure 3 H. pylori induces caspase-1 activation and IL-1β processing. 41igure 4 Different clinical isolated H. pylori strains induce IL-1β production. 42igure 5 H. pylori-induced IL-1β production in THP-1 cells is impaired in cagA deficient mutant. 43igure 7 H. pylori–induced caspase-1 cleavage and IL-1β production is impaired in cagA defect mutant. 46application/pdf725411 bytesapplication/pdfen-US胃幽門螺旋桿菌發炎體IL-1Helicobacter pyloriinflammasome[SDGs]SDG3http://ntur.lib.ntu.edu.tw/bitstream/246246/181794/1/ntu-97-R95449007-1.pdf