BACKGROUND/AIMS: Helicobacter pylori infection is linked to the development of gastric cancer. H. pylori-associated gastric inflammation is considered to be the first important step in the histogenesis of such neoplasia. However, studies that compare proteome of gastric mucosa infected with or without H. pylori are lacking. METHODS: We employed proteomics analysis on the endoscopic biopsy specimens of gastric mucosa obtained from two groups (30 cases): healthy subjects without H. pylori infection (15 cases), and gastritis patients with H. pylori infection (15 cases). The pooled proteins obtained from gastric mucosa infected with or without H. pylori were separated by two-dimensional gel electrophoresis and analyzed by a computer-aided program. The altered protein expressions were then identified by mass spectrometry and validated by Western blotting and immunohistochemistry. RESULTS: On mass spectrometry using MALDI TOF(TM) Analyzer, the up-regulation of Keratin 1, ezrin, adenosine triphosphate (ATP) synthase subunit alpha mitochondrial isoform c, Keratin type I cytoskeletal 19, and Keratin type I cytoskeletal 9 were identified; in contrast, 71 kd heat shock cognate protein, ATP synthase subunit alpha mitochondrial precursor, and annexin IV were down-regulated. Among them, membrane cytoskeleton linker ezrin was validated using Western blot and immunohistochemistry. CONCLUSIONS: Expression of ezrin was significantly different between the gastric mucosa with and without H. pylori infection. Therefore, ezrin could be considered a promising potential molecular marker for detecting H. pylori infection in gastric mucosa.
Blotting, Western ; Cytoskeletal Proteins/metabolism ; Down-Regulation ; Electrophoresis, Gel, Two-Dimensional ; Female ; Gastric Mucosa/*metabolism/microbiology ; Gastritis/complications/metabolism/pathology ; Gastroscopy ; Helicobacter Infections/complications/metabolism/*pathology ; *Helicobacter pylori ; Humans ; Immunohistochemistry ; Male ; Proteome/*analysis ; *Proteomics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Up-Regulation

OBJECTIVETo investigate whether Helicobacter pylori infection has any effects on the epithelial cell proliferation, apoptosis and P53 gene expression as well as its role in the pathogenesis of chronic gastritis.

METHODSSixty children with chronic gastritis were studied. All the children underwent upper digestive tract endoscopy and biopsy specimens were taken. Helicobacter pylori infection was determined with microscopic examination after Gimsa staining and the rapid urease test and 30 of the children were Helicobacter pylori positive and the other 30 were negative. The relation between the findings and cell proliferation was studied by immunostaining; the status of gastric apoptosis was tested by DNA fragmentation in situ using TdT-mediated dUTP biotin nick end labeling (TUNEL). Immunohistochemical method was used to detect the expression of P53 protein; CagA antibody was tested by Western blotting.

RESULTS(1) The proliferative index and apoptosis index in children with Helicobacter pylori infection with CagA positive gastritis were much higher than those of Helicobacter pylori negative gastritis patients [(11.56 +/- 4.21)% vs. (5.85 +/- 2.21)%, (10.58 +/- 5.31)% vs. (2.86 +/- 0.64)%, P < 0.01]. (2) The proliferative index and apoptosis index in 30 cases with Helicobacter pylori infection with CagA positive gastribis were much higher than 21 cases who were cured by effective drugs [(11.50 +/- 4.11)% vs. (3.74 +/- 2.30)%; (10.58 +/- 4.02)% vs. (3.74 +/- 2.30)%, P < 0.01]. (3) The expression of P53 protein in Helicobacter pylori with CagA positive gastritis children was much higher than that of Helicobacter pylori negative cases [(63% vs 16%), P < 0.1].

CONCLUSIONCagA positive Helicobacter pylori infection with gastritis improved gastric epithelial cell proliferation and apoptosis. The abnormal expression of P53 protein in gastric epithelium may play an important role in regulation of the processes.

Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; Apoptosis ; Bacterial Proteins ; immunology ; Biopsy ; Cell Proliferation ; Child ; Child, Preschool ; Epithelial Cells ; metabolism ; Female ; Gastric Mucosa ; pathology ; Gastritis ; complications ; pathology ; Helicobacter Infections ; complications ; pathology ; Helicobacter pylori ; Humans ; In Situ Nick-End Labeling ; Male ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo establish an animal model of gastric cancer by long-term infection of Helicobacter pylori (H.pylori) and to elucidate the pathogenesis by proteomics analysis.

METHODSFifty male Mongolian gerbils (4-5 week-old and weighted 60-100 g) were infected with H.pylori and the gastric tissues were obtained after the infection at 3, 6, 12 and 24 months. Histological changes were evaluated by H-E staining of the gastric tissue sections. Detection of H.pylori was performed by in-vitro culture of fresh gastric tissue samples, PCR amplification of H.pylori 16s rRNA and localization by silver staining. In addition, proteins extracted from gastric tissue samples were subjected to two-dimensional electrophoresis (2-DE) at various infection time points. Protein spots with increased quantity over the course of H.pylori infection were selected and analyzed by LC-MS/MS. Finally, differentially expressed proteins between human gastric cancer tissue samples and lymph nodes were analyzed by real-time RT-PCR.

RESULTSColonization of H.pylori was observed in gastric tissue of gerbils as early as 3 months after H.pylori infection, and persisted till 24 months. Pathological examination of infected animals showed various histological changes including acute gastritis, atrophic gastritis, intestinal metaplasia and gastric carcinoma. Seventy-eight differentially expressed proteins were identified by proteomics analysis, among which 36 proteins were up-regulated and 42 were down-regulated. Analyzed by LC-MS/MS, ten proteins were identified, including lactate dehydrogenase, ATP synthase, fatty acid-binding protein, COX5B, peroxiredoxin-4, peroxide reductase, transgelin, succinyl-CoA ligase, keratin and protein disulfide-isomerase A2, among which transgelin, ATP synthase and lactate dehydrogenase were highly expressed in human gastric carcinoma and lymph nodes.

CONCLUSIONSH.pylori infection induces the expression of transgelin, ATP synthase and lactate dehydrogenase, implying possible roles in the pathogenesis of gastric diseases including cancer.

Animals ; Disease Models, Animal ; Gastritis ; microbiology ; pathology ; Gerbillinae ; Helicobacter Infections ; complications ; metabolism ; Helicobacter pylori ; genetics ; Humans ; L-Lactate Dehydrogenase ; metabolism ; Male ; Metaplasia ; Microfilament Proteins ; metabolism ; Muscle Proteins ; metabolism ; Proteomics ; Proton-Translocating ATPases ; metabolism ; RNA, Ribosomal, 16S ; analysis ; Stomach Neoplasms ; metabolism ; microbiology ; Tandem Mass Spectrometry