| Background and aims.Helicobacter pylori(H.pylori) is a gram-negative bacterium that resides in the mucosa of the human stomach.It hasbeen generally accepted that colonization of the stomach by H.pylori is associated with the risk of different gastroduodenal diseasesincluding chronic gastritis,peptic ulcer,gastric cancer,and mucosassociatied lymphoid tissue lymphoma.Although the current recommended treatment regimens(bismuth salts or proton pump inhibitor in combination with two antibiotics) are effective,the high cost of medication and increasing occurrence of antibiotic-resistant strains hamper the efficacy of chemotherapeutic agents.So the use of vaccine immunization in the prevention and control of H.pylori infection may be a optical alternative.Animal studies have demonstrated that immunization with H.pylori whole-cell extracts or purified components plus a mucosal adjuvant can be developed for the prevention as well as the treatment of H.pylori infection.However the protective mechanisms of vaccination is not fully understood,which hinder the reseach of more effective H.pylori vaccines.So our reseach aims to investigate the effects of the oral vaccine containing Helicobacter pylori (H.pylori) sonicate plus cholera toxin B subunits(CTB) in the prevention of H.pylori infection in mice,and study the protective immune response induced by the vaccine .Methods: (1)H.pylori were cultured and identified:The syndney stain 1 of H.pylori(SS1),which was kindly provided by Sun Yat-Sat University of medical sciences,was grown on medium and incubated under mi 5croaerobic conditions(O25%, CO210%, N285%) at 37℃ for 2-3 days,H.pylori was identified by germ figure,rapid urease test,oxidase test and histology.(2)Preparation of H.pylori antigen:Cultures were centrifuged at 4℃,The pellet was washed in phosphate-buffered saline(PBS),and cells was disrupted by sonication.After centrifugation,the supernatant was collected,the protein concentration was determined,and the supernatant was frozen at –20℃ until use.(3)Vaccination and challenge of mice: Specific-pathogen-free 6-week-old female BALB/C mice randomly divided into four groups. Each group had 20 mice .Mice were orally immunized by H.pylori sonicate(2mg) plus CTB alone or PBS, respectively ,once weekly for four weeks.Four weeks afterthe last immunization ,all animals were challenged by alive H.pylori(1ⅹ108 organisms) two times with a four-day period.(4)Test figure: Four weeks after the last immunization and challenge,mice were sacrificed.Serum was collected to test anti-H.pylori IgA, IgG1 and IgG2a and the change of the pathology by culture.The stomach was isolated for examination for H.pylori histological examination,and determination of secretory anti-H.pylori IgA.Results:(1)Protective rate of the immunized group was 73.33%,whilethe controls were all infected(P<0.001). In protected mice,however,severe gastritis characterized by marked infiltration of inflammation mononuclear cell was noted at four weeks after challenge,compared with the gastritis seen in control groups(P<0.01). (2) Before challege the levels of H.pylori specific IgA, IgG1 and IgG2a in serum were higher than those of control mice(P<0.01),so was the labeling index for IgA-positive lumina of pyloric glands.After challenge,higher levels of antibodies were noted in these mice,the immunized group had a significant higher level of IgG2a than that of IgG1, so IgG1/IgG2a ratio decreased from 0.67 to 0.59. 6Conclusions:The oral vaccine containing H.pylori sonicate plus CTBis effective in preventing from H.pylori infection in mice ,More severer gastritis,however ,can be induced.(2)Anti-H.pylori IgA antibody plays a primary role in protection against H.pylori infection .(3)The vaccine can induce Th1-and Th2-mediated protective immune response in mice,and the effect of Th1-induced response is predominant.
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