Preliminary Study Of Immune Response And Mechanism Of DC In Mice Induced By A Mimic Peptide From HCV HVR1

This reseach was established on the study of HCV strains(Ⅱ/1b,Ⅲ/2a) envelope protein E2 hypervariable regions 1(HVR1) sequence variation rule and synthesis HCV HVR1 momoepitope according to the conservation site.As specialized antigen-presenting cells, dendritic cells(DC) played a pivotal role in immune responses.DC were commonly considered to be terminally differentiated cells derived from hematopoietic progenitors or monocytes.Our research used BALB/C mouse as the study object and investigated the immune response and its mechanism of DC induced by a polypeptide(7P).The reseach was divided to three sections.(一) To establish a method of cultivation and purification of dendritic cells from mouse bone marrow in vitro and observe theirmorphology.Mouse bone marrow cell suspensions were prepared with aseptic technique,then monocytes were isolated and induced in vitro by both granulocyte macrophagecolony-stimulating factor(GM-CSF) and interleukin 4(IL-4).The cell morphology and phenotype were analyzed.The percentage of the cultured DCs was 80% with typical morphology of DCs under light microscope.The CD11c~+ expression rate of DC cultured in vitro was more than 80%.A large number of DCs from mouse bone marrow in vitro exhibited typical morphology of DC,which might be an experimental material for further study on function,marker and antitumor role of DCs in vitro.(二)To study DC cells function of the mouse immunized by synthesis peptide.After subcutaneously immunization for ten days,the mice splenocytes were harvested. CD4~+Tand CD8~+Tcells were separated from splenocytes by cell sorting technology.Then CD4~+Tcells,CD8~+Tcells and DC cells were incubated for a day.The supernatants were deteced by cytometrie bead array Protein arraysystem(CBA).Compared with control group,the levels of IFN-γ,TNF-α,IL-10 and IL-6 secreted by CD8~+Tcell were increased in experiment group,the level of TNF-αsecreted by CD4~+Tcell was lower and the levels of IL-5,IL-2 and IFN-r were higher in experiment group,the differences were statistically significant between both group.In conclusion,the peptide could induce cell-mediated immune response through CD8~+ T and Th1 cells.(三) To study the mechanism of immune response induced by synthesis peptideIn order to analyze the mechanism of immune response induced by synthesized peptide, the chip of affymetrix mouse genome 430 2.0 assay was applied.The differences in expression levels of genes were analyzed with cluster analysis and bioinformatics assay. Compared with control group,the mRNA levels of Gp1bb,Hp,F13a1,F2r12,Gp5,F10 and Vwf were higher,and those of Tnfrsf13c,Syk,Tcrb-V13,Fgd4,Igh-6,Pde4b, Socs3,Jak1 and Tnfsf13b were lower in experiment group.At the same time,the mRNA levels of syk,Jak and Tnfsf13b were lower than the control by RT-PCR assay,but those of IFN-γ,IL-2 and IL-10 were higher.The protein expression level of Syk was lower than the control by Western Blot assay,but that of IFN-γwas higher.In conclusion,the peptide could induce cell-mediated immune response through CD8+ T and Th1 cells,while the inflammation was inhibited by some factors.