Impact Of Echinococcus Granulosus Recombinant Antigens Of Eg.ferrtin And Eg10on Mouse Bone Marrow-derived Dendritic Cells

Objective:1.To observe the changes of the mouse bone marrow-deriveddendritic cells(BMDC) stimulated by Echinococcus granulosus recombinant antigenEg.ferrtin which have protection and Eg10which have no protection in vitro, Bystudying the differences in their morphology, surface molecule expression andimmunological function of BMDC stimulated by different antigens in order to explorethe differences of immune mechanisms produced by Eg.ferrtin and Eg10.2.Its also inorder to further explore the basic theory of parasitic infections, for us to find newtherapeutic targets to provide direction.Methods:1. obtain the soluble form of Echinococcus granulosus recombinant antigenEg.ferritin and Eg10：recombinant plasmid Eg10/pET-28a and Eg.ferritin/pET-28awere induced under IPTG to a final concentration of0.2mmol/L to1.0mmol/L anddifferent temperature of25℃,28℃,30℃,37℃, the optimal conditions for proteinexpression was explored; using GSSH refolding solution for inclusion proteinrefolding conditions were optimized to observe protein refolding conditions.2. Co-culture of BMDC with Echinococcus granulosus recombinant antigen Eg.ferrtinand Eg10in vitro:Bone marrow-derived dendritic cells were generated from bonemarrow cells of mice with CD34+cells as a precursor cells cultured with recombinantmurine granulocyte macrophage colony-stimulating factor (rmGM-CSF) andrecombinant mouse interleukin-4(rmIL-4)，the cell culture medium were changed by the next day in half。After culturing for8days,we obtained the BMDC.The BMDC were divided into four groups: one group was added Echinococcusgranulosus recombinant antigen Eg.ferrtin, one group was added Echinococcusgranulosus recombinant antigen Eg10, the two groups were used0.1ug/ml,1ug/ml,10ug/ml three concentrations of antigen and6h,20h,48h different stimulation time toselect the best concentration and stimulation stimulation time; one group join LPS(50ng/ul) as a positive control group; one group do not add any substance used as acontrol group.3. morphological observation: the external structure and ultrastructure of eachBMDC groups were examined by SEM and TEM,comparing the number of glitchesand the changes in the ultrastructure of each group.4. Detection of the expression of surface molecules of BMDC: the surfacemolecules of BMDC of each group were analyzed by flow cytometry(FCM),thesurface molecules of BMDC were detected through MHCⅡmolecules andcostimulatory molecules (CD40, CD80, CD86).5. The influence of BMDC on T cell proliferation:The four groups of BMDCwere cocultured with the allogeneic T lymphocyte cells. The proliferation of Tlymphocyte cells in mixed lymphocyte reaction(MLR)to compare the difference ofeach BMDC group on T cell proliferation.6. Detection the level of Cytokines secreted by BMDC:To collect culturesupernatant of four groups, interleukin-6(IL-6)、interleukin-10(IL-10)、interleukin-12(IL-12)、TNF-α of culture supematant by BMDC were detected with enzymelinked immunosorbent assay(ELISA) to evaluate the exprssion levels betweendifferent groups.Results:1. recombinant expression plasmid Eg10/pET-28a and Eg.ferritin/pET-28a were induced to express with the final concentration of IPTG of0.2mmol/L to1.0mmol/L and different temperature of25℃,28℃,30℃,37℃.The form of theEchinococcus granulosus recombinant antigen Eg.ferrtin and Eg10were still inclusionbodies, we get considerable soluble form of Echinococcus granulosus recombinantantigen Eg.ferrtin and Eg10by using GSSH refolding solution.2. we established successfully the method to cultue the BMDC in vitro.About1-2×107dendritic cells at above70%purity can be amplified from bone marrow oftwo mice.3. Eg.ferrtin recombinant antigens can stimulate the BMDC mature in vitro.TheBMDC stimulated by Eg.ferrtin with the typical morphology of mDC can stimulateallogeneic T lymphocyte cells proliferation, which high expressed MHC-Ⅱ moleculesand costimulatory molecules.It also highly expressed the Th1cytokines of IL-12andTNF-α，compared with the control group,the difference was significant(P<0.01).4. Eg10recombinant antigens can not stimulate the BMDC mature in vitro. TheBMDC stimulated by Eg10induced poor allogeneic T lymphocyte cellsproliferation,which intermediately expressed MHC-Ⅱ molecules and lowly expressedcostimulatory molecules.Th2cytokines of IL-6were highly expressed,compared withthe control group,there was significant(P<0.05),the expression of IL-10comparedwith the control group was significantly higher(P<0.01).Conclusion:1.Eg.ferrtin recombinant antigens can stimulate the BMDC mature. The BMDCstimulated by Eg.ferrtin can highly stimulate allogeneic T lymphocyte cellsproliferation and differentiation, which highly expressed the Th1cytokines of IL-12and TNF-α.It is suggested that Eg.ferrtin recombinant antigens may cause the body toproduce an immune response mediated by Th1type cells, so that by the originalpathogen destruction, to remove the invading pathogens and protective role of the body.2.Eg10recombinant antigens can not stimulate the BMDC mature. The BMDCstimulated by Eg10induced poor allogeneic T lymphocyte cells proliferation,whichTh2cytokines of IL-6and IL-10were highly expressed,suggesting that Eg10recombinant antigens may cause the body to produce an immune response mediatedby Th2type cells induced immune suppression effect, thus contributing to the survivalof pathogens.