CD4～+ T Lymphocyte Reaction In HP Infected C57BL/6 Mice And Effects Of Anti-HP Lactobacillus Acidophilus On HP Infection

PartΙCD4~+T helper cell reaction in C57BL/6 mice with HP infectiveObjective: To analyze CD4~+ T helper reaction and offer bases to select immunoregulant by establishing Helicobacter pylori infected animal model. Method: To divide 16 C57BL/6 mice into two groups randomly and feed with HP SS1 or PBS. Four weeks later execute mice by cervical vertebra dislocation method and get stomach tissue to carry out Warthin-Starry and HE stain, then evaluate stomach mucosa inflammation and HP density according to Sydney system. Detect reproductive activity of spleen lymphocyte cell under stimulation of non-specificity antigen and HP specificity antigen. To employ fluorescein isothiocyanate labeling CD4 cell and phycoerythrin labeling interleukin-4 and Interferon-gamma monoclonal antibody incubate with spleen lymphocyte and analyzed by flow cytometry. Results: The stomach tissue HP positive rate that were inoculated HP SS1 was 100%. HP locate on the surface of gastric membrane, gastric scrobiculus and intracavitary, and its surrounding immersed by lymphocyte and monocytes. While HP was not found in gastric membrane of the mice feed with PBS. Furthermore, no inflammatory cell infiltrate in gastric membrane. Cell reproductive activity of spleen lymphocyte of HP infective and non-infective mice increased with increased dimension of ConA and HP antigen. Lymphocyte reproductive activity is the strongest when density was 7.5μg/ml and 0.15mg/ml respectively. CD4~+ IFN-γ~+ and CD4~+ IL-4 + cell proportion were all increased in the HP infective group compared with non HP infective group. Especially CD4~+ IFN-γ~+ cell proportion increase the more, so CD4~+IFN-γ~+/CD4~+IL-4~+ rate increased compared with control group, p<0.05. Conclusion: The method that inoculated HP SS1 with feeding could induce SPF C57BL/6 mice gastric mucosa damage similar to human. Lymphocyte reproductive activity of spleen in HP infective C57BL/6 mice increased under stimulation of ConA and HP antigen compared with non- HP infective. The percentage of CD4~+ IFN-γ~+ cell was higher than CD4~+IL-4~+, suggest immune response mediated Th1 play an important role in chronic inflammatory reaction induced by HP infection.PartⅡInfluence on CD4~+ T helper cell reaction in C57BL/6 mice with HP infection treated and prevented by anti- HP Lactobacillus acidophilusObjective : To determine reaction of CD4~+ T helper and approach immunoregulation mechanism by observing prevention and treatment effect on HP infection by anti-HP Lactobacillus acidophilus. Method: To divide 24 C57BL/6 mice into three groups. Id est HP infection group, HP inoculation after feeding with anti-HP Lactobacillus acidophilus group, feeding with anti-HP Lactobacillus acidophilus after HP inoculation group. Four weeks later execute mice by cervical vertebra dislocation method and get stomach tissue to carry out Warthin-Starry and HE stain, then evaluate stomach mucosa inflammation and HP density according to Sydney system. To detect reproductive activity of spleen lymphocyte cell under the optimal concentration stimulation of non-specificity antigen and HP specificity antigen by MTT method. CD4~+T cell was analyzed by flow cytometry. Results: The stomach tissue HP positive rate that were inoculated HP after feeding with anti-HP Lactobacillus acidophilus was 0%,HP density and chronic inflammation score lower than HP infection group. HP positive rate was 100% in the group feed with anti-HP Lactobacillus acidophilus after inoculating HP, but HP density and chronic inflammation score lower than HP infection group. Reproductive activity of spleen Lymphocyte lowered significantly under stimulation of optimal concentration stimulation of non-specificity antigen and HP specificity antigen compared with HP infection group. While reproductive activity can't be inhibited in the group feed with anti-HP Lactobacillus acidophilus after inoculating HP. The proportion of CD4~+ IFN-γ~+ and CD4~+ IL-4~+ cell of spleen lower in the group inoculated HP after feeding with anti-HP Lactobacillus acidophilus compared with the group infected by HP, and so on the ratio of CD4~+ IFN-γ~+/CD4~+ IL-4~+, p<0.05. The proportion of CD4~+ IFN-γ~+ ,CD4~+ IL-4~+ cell and CD4~+ IFN-γ~+/CD4~+ IL-4~+ of spleen was similar in the group feed with anti-HP Lactobacillus acidophilus after inoculating HP and the group infected by HP. Conclusion: The method that feed with anti-HP Lactobacillus acidophilus could prevent infection induced inoculating HP; Apply anti-HP Lactobacillus acidophilus to treat HP infection could decrease HP density and chronic inflammation score. Spleen reproductive activity induced by HP infection can be inhibited by feeding with anti-HP Lactobacillus acidophilus in advance. In addition, cytokine by Th1 can also be inhibited. While reproductive activity can't be inhibited if feed with anti-HP Lactobacillus acidophilus after inoculating HP, and so on level of Th1 cytokine. immunomodulationPART III Influence on reaction of CD4~+ T helper of C57BL/6 mice with Helicobacter pylori infection treated long term by anti- HP Lactobacillus acidophilus fermented milkObjective : To determine reaction of CD4~+ T helper and approach immunoregulation mechanism by observing long term treatment effect on HP infection by anti-HP Lactobacillus acidophilus fermented milk. Method: To divide 72 C57BL/6 mice into three groups. The mice of three groups feed with anti-HP Lactobacillus acidophilus fermented milk, common fermented milk and PBS after inoculating HP. Execute mice by cervical vertebra dislocation method when treatment course was 3, 6 and 9 month and get stomach tissue to carry out Warthin-Starry and HE stain, then evaluate stomach mucosa inflammation and HP density according to Sydney system. To detect reproductive activity of spleen lymphocyte cell under the optimal concentration stimulation of HP antigen by MTT method. CD4~+T cell was analyzed by flow cytometry. Result: The eradication rate was 25% and 62.5% respectively when six and nine month,while no eradication arise in control group. HP density and chronic inflammation score lowered significantly compared with the other group when treatment course three month. Furthermore HP density and chronic inflammation score lowered more with prolong of treatment course. Reproductive activity of spleen lymphocyte was inhibited by treatment of anti- HP Lactobacillus acidophilus fermented milk. The inhibition was obviously in nine month. CD4~+ IFN-γ~+ cell was inhibited by feeding with anti-HP Lactobacillus acidophilus fermented milk to treat HP infection. The inhibition was obviously in nine month. Conclusion: Apply anti-HP Lactobacillus acidophilus(L4) fermented milk to treat HP infection could decrease HP density and chronic inflammation score, long term treatment could eradicate HP . Reproductive activity of spleen lymphocyte and level of CD4~+ IFN-γ~+ cell was inhibited by long term treatment of anti- HP Lactobacillus acidophilus fermented milk for HP infection compared with HP infection and common fermented milk group.