The Effect Of B7 Molecules On The Immune Responses To The Anti-caries DNA Vaccine

Dental caries are common infectious diseases,which severely affect the oral health of human.Through many years researches,our group has constructed one kind of targeted DNA vaccine which contains the targeted protein cytotoxic T lymphocyte antigen 4(CTLA-4)fragments and specific antigen GLU.And we have proved that this DNA vaccine is an effective way to prevent dental caries.But until now,there are still several questions remain to be solved,such as how does this DNA vaccine works,what influences will this vaccine bring to the type of immune response and what are the functions of CTLA-4,B7-1 and B7-2 molecules during the immune response initiated by the vaccine.To make these questions clear,we makes preliminary research and hope we can know the influences of B7-1 and B7-2 molecules to the type of immune response and the effect of this vaccine.Experiment 1 Research on the influences that knockout of B7 molecules bring to the effect of anti-caries DNA vaccineObjective:Use the B7-1 and B7-2 knockout mice to investigate the impacts of these two molecules on the effect of anti-caries DNA vaccine in vivo.Method:Immune the B7-1 KO,B7-2 KO,B7-1/2 KO and wild type(WT)mice through nasal cavity with the targeted anti-caries DNA vaccine.And then collect the saliva and serum samples of these mice at different time.ELISA detected the levels of specific antibody of saliva IgA and serum IgG.Results:In B7-1/2 KO group,when compared with the WT group,the contents of IgA in the saliva and IgG in the serum both went down in various degrees.And we found from the result that IgA in saliva had the highest content in the 6th week,and which was the 4th week in the WT group.In B7-1 KO group,when it compared with the WT group,the content of IgA also significantly went down,and the highest level of IgA were also appeared in 6th week.The levels of IgG in serum were lower than the WT group excluded the 6th week.In B7-2 KO group,the levels of IgA were only lower in 6th week,and the levels of IgG in serum were only lower in 10th week.Conclusion:B7-1 and B7-2 molecules can promote the product of specific antibody in saliva and serum after immune the targeted anti-caries DNA vaccine.And the role of B7-1 is stronger than B7-2 in this aspect.Experiment 2 Knockout of different B7 has different impacts on the types of immune response induced by this DNA vaccineObjective:To investigate the impacts of B7-1 and B7-2 on the effect of anti-caries DNA vaccine in vivo and the types of immune response.Method:Immune the B7-1 KO,B7-2 KO,B7-1/2 KO and wild type(WT)mice intramuscularly with the targeted anti-caries DNA vaccine.And then the spleens of these mice were removed to collect spleen cells.Stimulated these cells with GLU and 24 hours later,collected the supernatants.ELISA detected the levels of IFN-γ,IL-17,IL-10 in the supernatants.Results:In B7-2 KO group,when compared with the WT group,we could find that IFN-y had a higher content;So maybe B7-2 molecule could inhibit the secretion of IFN-y.In each gene knockout group we found the level of IL-17 had no obvious changes,which indicated that IL-17 had no connection with B7-1 and B7-2.In B7-1/2 KO group,the level of IL-10 had no obvious changes either,whereas it went up in some degree in the other two gene knockout groups.Conclusion:B7-2 molecules can inhibit the type of Thl immune response.And there are no obvious impact on the type of Th17 immune response of B7-1 and B7-2 molecules in vivo.Experiment 3 Researsh on the effect of B7 molecules on T lymphocyte proliferation and differentiationObjective:Use the B7-1 and B7-2 knockout mice to study the influences of B7 molecules on the activation of T cells.Method:Separated the femurs and tibias from all these groups.Then we got the bone marrow from these bones.Through appropriate culturation,we harvested dendritic cells(DCs).LPS was used to make sure that DCs became mature.Then we cultured T cells company with DCs.After the culturation,the supernatants were collected to detect the content of IFN-γ,IL-4 and IL-17 using the ELISA kit.And the cells were collected to get RNA for Quantitative real-time PCR(qPCR).We used the CCK8 kit to test the status of the proliferation of T cells.Results:The result of B7-1 KO group indicated that when it compared with the WT group,the IFN-y and T-bet in its supernatants and cells both went down,IL-4 and GATA-3 both went up in some degree.However the content of IL-17 and RORyt had no significant changes.The result ofB7-2 KO group indicated that when it compared with the WT group,the IFN-γ and T-bet in its supernatants and cells both went up,whereas the IL-4,IL-17,GATA-3 and RORyt all went down in some degree.The result of B7-1/2 KO group indicated that when it compared with the WT group,the changes of all these factors were just between the other two groups.And the proliferation test indicated that the differences between each two groups had no statistical significance.Conclusion:B7-1 and B7-2 molecules have different influences in T lymphocytes.The former one can promote the native cells turn into Thl cells,block them turn into Th2 cells,and has no obvious connection with the differentiation of Th17 cells.While B7-2 can promote the native cells turn into Th2 cells and Th17 cells,block them turn into Thl cells.In addition,the B7-1 and B7-2 molecules have no influences in the proliferation.