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The Preliminary Study of the Biological Functions of Plasticity Gene jhp947

Author: LinLiPing
Tutor: ZuoFeiFei
School: Fujian Medical
Course: Pathogen Biology
Keywords: Helicobacter pylori genes of plasticity region jhp947 gene function
CLC: R378
Type: Master's thesis
Year: 2009
Downloads: 31
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Abstract


Background and Objectives:Helicobacter pylori (Hp) is a gram-negative, microaerophilic and spiral-shaped microorganism that colonizes the human astric mucosa. It is well known that Hp is recognized as the etiological agent of gastric diseases such as chronic atrophic gastritis and peptic ulcers, and the infection with Hp is associated with the development of gastric carcinoma and mucosa-associated lymphoid tissue lymphoma. However, little is known about the molecular mechanisms of the pathogenesis induced by Hp and the fundamental reasons of diversity of infection outcomes. Recently,pepole begant pay attention to the plasticity region of Hp for seeking the pathogenic gene The Helicobacter pylori plasticity region locus jhp947 is one of the important virulent factors of Hp. Recent studies have indicated jhp947 is associated with peptic ulcer and gastric carcinoma. In the present study. firstly the virulent effects on host cell of jhp947 were preliminarily assessed via comparation of biological characteristics between wild-type strain J99 and isogenic mutant J99-947△,and of the in vitro effects on the proliferation and apoptosis of the gastric epithelium cell GES-1 cells induced by two strains. Then, Enzyme-Linked Immunosorbnent Assay(ELLISA) was used to screen and identify the differential secretion of cytokines interleukin(IL)-8 and IL-12. This study provided the important experimental evidences for clarifying the roles that jhp947 plays in the pathogenesis induced by Hp and for elucidating the association between jhp947 and gastric cancer.Methods:1 Analysis of the virulence activity of the jhp947-knocked out mutant 1.1 The activities of urease of wild-type strain J99 and isogenic mutant J99-947△were assayed by the urease-testing reagents.1.2 The stab agar test was used to observed the motility of wild-type strains and isogenic mutants.1.3 The different adherent effects on human gastric epithelium cell GES-1between wild-type strains and isogenic mutants were investigated by adherence experiments.1.4 The toxic effects of vacuolating toxin of wild-type strains and isogenic mutants on GES-1 cells were evaluated by both light microscopy and neutral red uptake (NRU) assay.2 Effects of the jhp947-knocked out mutant on the proliferation and apoptosis of GES-1 cells.2.1 GES-1 cells were co-cultured with the wild-type strains and the isogenic mutants at a bacteria:cell concentration of 100:1, respectively.2.2 The cell phenotype was observed at 6,12,24 and 48h after co-culturing.2.3 The cellular proliferation was examined by Cell Counting Kit-8 (CCK-8) method at time points of 24h after co-culturing.2.4 The induction of apoptosis of cells co-cultured with H.pylori for 24h was determined by flow cytometry.3 Effects of the jhp947-knocked out mutant on the secretion of cytokines interleukin(IL)-8 and IL-12.3.1 AGS were cocultured respectively with the wild-type strains and the mutants for 24h and the suspension were collected measurement IL-8 by ELISA.3.2 Generation and identification of dendritic cells(DCs) from human peripheral blood3.3 Immature DCs were cocultured respectively with the wild-type strains and the mutants for 48h and the suspension were collected measurement IL-12 by ELISA.3.4 THP-1 cell were cocultured respectively with the wild-type strains and the mutants for 24h and the suspension were collected measurement IL-12 by ELISA. Results:1. Analysis of the virulence activity of the jhp947-knocked out mutant of J99.It was showed that J99 and wild-type strains J99-947△could lead to remarkably color changes of the unease-testing reagents in a very short time. The OD490nm values of unease reagents treated by Hp investigated that OD values of J99-947△at different time points from 5 to 30 minutes were similar to those of J99. which indicated that there was no difference in the activity of unease between these two experimental groups.In the stab agar test, J99 group formed a similar swarming zone with J99-947△group after at least 3 days of incubation in 0.3% motility agar, which indicated that there was no remarkably difference in the motility between these two experimental groups. And it was not found that ablation of jhp947 gene had any apparent effect on vacuolating cytotoxin activity and adherence of Hp.2. The effects of the jhp947-knocked out mutant on the proliferation and apoptosis of GES-1 cells.The changes of cell morphology were observed when GES-1 cells were cocultured with Hp for 6 hours, and cell scatterring and vacuolate degeneration were presented after cocultured 48 hours in both groups.The CCK-8 assay showed an increasing tendency in the state of low infection with J99 and J99-947△. While ,there was no remarkably difference in cellular proliferation between these two experimental groups. As demonstrated by flow cytometry, the apoptotic rates in both groups treated by J99 (15.3%) and J99-947△(14.2%) were significantly higher than that of control group(1.7%), but there was no difference between these two experimental groups.3. Effects of the jhp947-knocked out mutant on the secretion of cytokines interleukin(IL)-8 and IL-12.Compared with control group,significantly higher levels of IL-8 were detected in AGS cells cocultured with J99 and J99-947△(p<0.05), respectively. While, there was no remarkably difference between these two experimental groups(p>0.05). Induce DC from PBMC of healthy men with GM-CSF and IL-4. This kind of DC is immature DC by analyzing the functions. Compared with control group, significantly higher levels of IL-12 were detected in DC and THP-1 cells cocultured respectively with J99 and J99-947△(p<0.05). And IL-12 production of DC and THP-1 cells inducing by J99-947△were significantly lower than those of J99, which indicated that the Hp plasticity region locus jhp947 is associated with IL-12 production in monocyte cells (p<0.05).Conclusions:1. jhp947 gene knocked-out had no effect on the virulence activity of Hp. This includes: the activity of unease、the motility、vacuolating cytotoxin activity and adherence of Hp.2. There was no significant difference between J99 and J99-947△they effect on the proliferation and apoptosis of GES-1 cells.3. It was firstly showed that jhp947 gene may have some influence on the IL-12 production of DC and THP-1 cells inducing by Hp, jhp947 gene knocked-out could reduce the production of IL-12.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Pathogenic bacteria
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