Detection Of Helicobacter Pylori DNA In Gastric Epithelial Cells By In Situ Hybridization

Background Gastric cancer is the second most common fatal malignancy in the world and is the cause of more than 750 000 deaths annually. Human gastric carcinogenesis is a multistep and multifactorial process. The sequential stages of gastric carcinogenesis of human model are chronic superficial gastritis, atrophy, intestinal metaplasia, dysplasia and cancer. Helicobacter pylon infection may play a causative role at the early phases of the chain. Three separate groups from Japan elucidated that long-term infection with H.pylori induces adenocarcinoma in Mongolian gerbils. The role of H.pylori in gastric carcinogenesis is supported almost exclusively by epidemiological data and prospective histopathological studies. So far the mechanism of H.pyloni carcinogenesis has not been illuminated. Some microorganisms, especially hepatitis B virus, Epstein-Barr virus and so on, are known as biological carcinogens, and integration is the most important mechanism of carcinogenesis. Parsonnet supposed that H.pyloni have similar mechanism of virus carcinogenesis, viz H.pylori DNA integrate in gastric epithelial cells genome, which may induce transformation or malignancy of normal cell. However, there is no reliable evidence of integration of H.pyloni DNA in the human genomes so far. H.pyloni DNA must invade gastric ~5IJ( epithelial cells at first, and exists chronically in gastric epithelial cells in an unknown manner before integration. The aims of our study is to investigate whether H.pylori DNA is able to invade gastric epithelial cells by GenPoint catalyzed signal amplification system for in situ hybridization, and simuItane,J detect antigens of Hpylori in gastric mucosa by immunohistochemical technique in patients with H.pylori infection. Materials and Methods Clinical data According to H.py/ori scientific research criterion, 112 patients, including 28 H.pylori negative and 84 H.pylori positive, were collected. There are 24 cases with chronic superficial gastritis, 25 cases with precancerous changes and 35 cases with gastric cancer among H.pylori positive group, while 10, 16 and 2 cases, respectively among H.pylori negative group. lmmunokistochemical technique After deparaffination and rehydration of the specimens, H.pylori antigen was detected by Utrasensitive SAP kit (Maxim Biotech, mc, USA) as suggested by manufacturer. The first antibody is rabbit anti Helicobacter pylon (DAKO, Denmark). The sections were counterstained with Nuclear Fast Red (Maxim Biotech, mc, USA) and examined under a light microscope. Negative controls were carried out in the absence of the first antibody. In situ hybridization The size of biotinylated Long DNA Probe for H.pyloni M-r-26,000 Protein Gene (Maxim Biotech, mc, USA.) is 303 base pair. Sum sections were coated single well slides and hot plated for 12 hours before dewaxing and rehydration through graded alcohols to water. The sections were treated sequentially with 0.2% Triton-X100 for 15 minutes, 0.02M HC1 for 10 minutes and digested with 1% pepsin (Sigma, USA) for 15 minutes, 2Oug/ml proteinase K (Meack, Germany) for 30 minutes. The enzyme was inactivated by treatment with 0.2% glycine in PBS for 5 minutes. Then the sections were incubated sequent...