| Summary: | 碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系 === 101 === Helicobacter pylori (H. pylori) is a Gram-negative microaerophilic bacterium found in the stomach. More than half of the world population is infected. H. pylori mainly infects gastric epithelial cells and have been associated with gastritis, ulcer, and gastric cancer. Among the virulence factors of H. pylori, CagA is one of the factors that can only be found in H. pylori and is correlated with occurrence of the diseases. CagA is delivered into the host cell through type IV secretion system in which CagE plays an essential role. Recently, H. pylori infection has been found to upregulate osteopontin expression in infected patients. Osteopontin (OPN) is a secretory extracellular matrix protein. Earlier studies indicated that OPN can promote migration, invasion, and tumor progression. It is overexpressed in many cancers, including gastric cancer, as well as gastritis and ulcer. In addition, tumor associated trypsin inhibitor (TATI), like OPN, is highly expressed in gastric diseases We examine if H. pylori could enhance OPN expression in vitro and the mechanism behind this induction, as well as the relationship between TATI and H. pylori. Our results showed that infection of AGS cells with Western and clinical strains of wild type H. pylori would enhance cellular OPN protein level significantly when analyzed with western blot; whereas infection with cagA and cagE mutant strains did not change OPN protein level. Investigation of the mRNA expression with qRT-PCR revealed that infection with either wild type or mutant strains of bacteria did not affect OPN or TATI mRNA expression. Transfection experiment confirmed that CagA could induce cellular OPN protein expression. We postulated that the increase in OPN is probably due to one or more of the following, 1) increase in translation, 2) inhibition of secretion, and 3) inhibition of degradation. Co-treatment of H. pylori with translational inhibitor, NaF, demonstrated that OPN protein induction was not regulated through OPN mRNA translation. Treatment with Exo1, a secretory pathway inhibitor, showed that OPN is secreted through classical constitutional pathway; treatments with degradation pathway inhibitors, chloroquine and MG132 demonstrated that OPN is degraded through lysosomal pathway. In addition, OPN in the medium was collected after infection with H. pylori. Analysis of secretory OPN with western blot imparted that H. pylori infection inhibited OPN secretion. In conclusion, virulent factor CagA from H. pylori could induce OPN protein expression by inhibiting OPN secretion. H. pylori induced OPN might mediate development of gastric cancer and tumor progression, and formation of hummingbird phenotype as well; thus, OPN can serve as candidate biomarker for H. pylori infection and early detection of gastric cancer.
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