| Objective:To investigate stimulation mature DCs (Dendritic cells) with recombinant Eg14-3-3(rEg14-3-3) and EgmMDH for antigen-presenting to activate and promote differentiation of naive CD4+T cells during infection of Echinococcos granulosus, we performed this study for description interplay between host immune response and E.granulosus infection.Method:1. Obtaining antigens:rEg14-3-3and rEgmMDH were expressed in plasmids of BL21, purified by nickel chelate affinity chromatography. And rEgmMDH was refolded to obtain a soluble antigen. Hydatid fluid (HF) and protoscoleces were obtained from E. granulosus cysts in patients with hydatid disease in aseptic conditions. Protoscoleces was ground in liquid nitrogen and sonication to obtain a soluble antigen.2. Preparation and cultivation of bone-marrow-derived dendritic cells (BMDCs):Normal mice bone marrow cells were isolated and culture in complete RPMI1640medium supplemented with IL-4and GM-CSF, then after7days BMDC were obtained.3.BMDCs stimulated with different Eg-antigens or lipopolysaccharide (LPS):Immature BMDC was stimulated respectively by LPS, rEg14-3-3, rEgmMDH, soluble protoscoleces antigen and HF for48hours. We observed the morphology of DC by scanning electron microscope, and then detected expression levels of cell-surface markers (CD40, CD80, CD86, MHC class Ⅱ) and polarization factor (IL-12and CCL2) by flow cytometry.4. Mixed lymphocyte reaction (MLR) and Fluorescence Activated Cell Sorter (FACS): Single cell suspensions of splenocytes were prepared by mincing the mouse spleens, and then CD4+T cells were isolated by the negative selection procedure of magnetic activated cell sorting. CD4+T cells were co-cultured with the DC pulsed with Eg-antigens or LPS for5days at the ratio of1:5. The proportion of Th1, Th2,Th17and Treg cells were detected by flow cytometry.Results:1. The high purified soluble recombinant antigens were harvested after the rEg14-3-3and rEgmMDH protein were expressed. Hydatid fluid (HF) and a soluble antigen of protoscoleces were obtained.2.①Results of scanning electron microscope showed that dendritic protrusions were differentiated when DC cells were stimulated by LPS, rEg14-3-3, rEgmMDH, protoscoleces extracts and HF.②Results of Flow cytometry showed that LPS, rEg14-3-3, rEgmMDH, soluble protoscoleces antigen and HF significantly increased the percentage of CDllc+MHC Ⅱ+cells and CD11c+CD40+cells compared with PBS(P<0.05). rEg14-3-3, LPS and protoscoleces extracts significantly increased the percentage of CD11c+CD80+cells and CD11c+CD86+cells compared with PBS(P<0.05). Results showed that no statistically significant differences were recorded in CD80expression between BMDC stimulated with rEgmMDH and those stimulated with PBS, or CD86expression between BMDC stimulated with HF and those stimulated with PBS (P>0.05).③Compared rEg14-3-3-stimulated BMDC with rEgmMDH-stimulated, analysis of the mean percentage showed that rEg14-3-3significantly increased the upregulation of MHC Ⅱ molecules, CD80and CD86expression (P<0.05).④The production of IL-12was a significant increase when BMDC were stimulated with LPS and rEgmMDH (P<0.05). A moderate increase in CCL2expression could be detected when BMDC were stimulated with rEg14-3-3and HF (P<0.05).3①Compared with PBS, the results of MLR showed that CD3+CD4+IFN-γ+T lymphotes were significantly increased in BMDC-LPS-stimulated CD4+T lymphotes after5days of culture, and A moderate increase in BMDC-rEg14-3-3-stimulated cultures or BMDC -rEgmMDH-stimulated cultures(P<0.05). But there were no statistically significant differences between BMDC-rEg14-3-3-stimulated cultures and BMDC-rEgmMDH-stimulated cultures(P>0.05).②CD3+CD4+IL-4+T lymphotes induced by BMDC pulsed with rEg14-3-3or HF were significantly increased compared with PBS(P<0.05).③The expression of CD3+CD4+IL-17+cell population were highest in CD4+T lymphotes from BMDC-rEgmMDH-stimulated cultures(P<0.05).④Compared with PBS,CD4+CD25+Foxp3+Treg cell population were significantly increased in BMDC-rEgmMDH-stimulated CD4+T lymphotes after5days of culture, as well as BMDC-HF-stimulated cultures or BMDC-protoscoleces extracts-stimulated cultures(P<0.05). A moderate increase in BMDC-LPS-stimulated cultures (P<0.05).Conclusion:1. Compared with the antigen rEgmMDH, mature DC was induced efficiently by rEg14-3-3.2. In vitro, experiments showed that the significantly higher levels of Thl and Th2cells are observed under rEg14-3-3induction, however, the differentiation of Th17and Treg cells in response to rEgmMDH stimulation is significantly more than that in response to rEg14-3-3stimulation. E.granulosus antigen-stimulating DCs possess to induce cellular immune response by stimulating CD4+T lymphotes, which give a strong evidence that DC play a crucial role in of different protective immunity interact with DC, and maybe play a role in E. granulosus infection. |
PDF Full Text Request