| Objective: Follicular helper T cells, Tfh cells) is a novel class of T cell subsets, Tfh cellsplay very important role B cell development, germinal center formation, antibody class switching,and establish with-term humoral immunity. In this study, we set up the specific expression ofFMDV VP1gene recombinant adenovirus (rAd5VP1) mice model, of FMDV VP1generecombinant adenovirus (rAd5VP1) immunized mice, which the mice were immunized withrAd5VP1, following the succeful model were set up, we were trying to look at the mice wereobserved for follicular helper T cells (T follicular helper cells, Tfh) differentiation, germinalcenter (GC) formation, Tfh cells transcription factor BCL-6mRNA expression level and theTfh cell cytokine secretion of IL-21levels, and explore the role of recombinant adenovirusvaccine during responses on the foot-and-mouth disease.Methods:6-week-old BALB/c mice were randomly divided into two groups of10.Experimental group were immunized with rAd5VP1, control group was injected with PBS.1. Day7after immunization, the mice spleen, mononuclear cells From the mice spleenisolated, Tfh cells (CD4~+CXCR5+PD-1~+) germinal center B cells (B220+GL-7+) detected byflow cytometry (FCM),the date was analyzied by.2. Day7after immunization, the mononuclear cells from the mouse spleen were isolated,takethe mouse spleen mononuclear cells, the transcription factor BCL-6mRNA of Tfh cells weremeasured by real-time RT-PCR.3. Day7after immunization, the mice was sectioned by routine paraffin techniques andstained by FITC-lectin (PNA), the routinely paraffin sections, FITC-lectin (PNA) staining,using microscopy to observe the formation of germinal centers.4. Day7after immunization, serum was collected from peripheral blood and the cytokinesinterleukin-21(interleukin-21, IL-21) was measured by by enzyme-linked immunosorbent assay (ELISA) kit.Results:1. On day7, from experimental group the data showed that flow cytometry results showed thatthe number of Tfh cells (CXCR5~+CD4~+) CXCR5~+of CD4~+cell ratio (4.05±0.10)significantlyhigher than the PBS control group (2.14±0.29), P=0.007.**P <0.01; The number of Tfhcells (CXCR5~+PD-1~+CD4~+) ratio (1.17±0.17) was significantly higher than the PBS thecontrol group (0.69±0.17), P=0.03.*P <0.05.2. On day7, the data showed that the number of the GL-7+B220+cell ratio (3.66±0.53) wassignificantly higher than the PBS control group (1.74±0.05), P=0.02.*P <0.05.3On day7, from experimental group the data showed that expression levels of BCL-6mRNA CT value by real-time RT-PCR, for getting2-ΔΔCT value, further statistical analysisof expression levels rAd5VP1group is2.39times than the amount of expression of the PBSgroup. P=0.001.**P <0.01.4. On day7, from experimental group the data showed that serum cytokines interleukin-21byenzyme-linked immunosorbent assay (ELISA) kit.after separation of the serum ELISA kit.The experimental group of IL-21expression levels (63.68±13.15, pg/ml) was significantlyhigher than the PBS control group (4.65±2.36pg/ml), P=0.02*P <0.05.5. On day7, from experimental group the data showed that microscopy to observe theformation of germinal centers by FITC-PNA staining. using Image J software to get averagefluorescent density values and know rAd5VP1group expression levels (12.54±1.77) wassignificantly higher than the PBS control group (6.71±0.55), P=0.001**P <0.01.Conclusions: We found that the Tfh cells can be induced by immunization of rAd5VP1on day7, meanwhile, the formation of germinal center can be enhanced and the differentiationof B cells can be promoted. can induce differentiation of Tfh cells, promote the formation ofgerminal center and promote the differentiation of B cells, The mRNA of Bcl-6also increased,the secretion of IL-21was boosted. Taking together, we conclude that Tfh cells and effectormolecules have played a very important role, for the subsequent vaccine adjuvant vaccineresearch, development direction, enhance the immune effect of vaccine provides theory basis. |
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