ABSTRACT
Objective:To investigate the immunomodulation of CCK8 on the Coxsackievirus B ( CVB )-attacked human peripheral blood plasmacytoid dendritic cells(pDC). Methods:Peripheral blood mononuclear cells of healthy volunteers were separated by Ficoll-Hypaque gradient density centrifugation. The pDC was separated and divided into five groups,which were the control group, CVB attacked group,the group of CCK8 treated after CVB attack,the group of PGE2 treated after CVB attack and the group of CCK8+PGE2 treated after CVB attack. 100-time TCID50 of CVB was applied for the attack on pDC. Real-time PCR and Immunofluorescence technique were employed to detect the expression of CCK1R/CCK2R mRNA and protein. Then,the expression levels of costimulatory molecules such as CD80,CD86,HLA-DR ligand,and the chemokine receptor CCR7 were evaluated by Flow Cytometry Analysis. The supernatants of pDCs were collected, and the content of IFN-α was determined by Enzyme-linked Immunosorbent Assay. Results:CCK1R and CCK2R were co-expressed in human peripheral blood pDC,and both were significantly upregulated after CVB attack in vitro. Expression of CD80,CD86,HLA-DR and IFN-α were decreased in the CVB+CCK8 group compared with the CVB group,which suggested that CCK8 may reduce the CVB activation of pDC. Whereas expression of CD80,CD86,HLA-DR,CCR7 and IFN-α were increased in the CVB+PGE2 group compared with the CVB group,which suggested that PGE2 may increase the CVB activation of pDC in vitro. Conclusion:CCK8 repressed the CVB-attacked pDC,while PGE2 activated the CVB-attacked pDC.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the presence of interactions between DNAJB13 and HK1.</p><p><b>METHODS</b>The open reading frame of Dnajb13 gene was amplified from mouse testis cDNA by PCR. The PCR products were then inserted into pGEX-4T-1 vector after double digestion and identified by sequencing. The recombinant plasmids were transformated into competent DH5a cells, and the fusion protein was expressed with IPTG induction. SDS-PAGE Coomassie brilliant blue staining and Western blot analysis were used to detect the fusion protein expression. The protein precipitated by GST-DNAJB13 in GST pull down assay was detected by Western blotting.</p><p><b>RESULTS</b>The recombinant plasmid pGEX-4T-1-Dnajb13 was successfully constructed and verified. E.coli transformed with the recombinant plasmid expressed abundant fusion protein. GST pull down assay showed interactions between DNAJB13 and HK1.</p><p><b>CONCLUSION</b>DNAJB13 interacts with HK1 in mouse testis and probably participates in spermatogenesis and the regulation of sperm motility.</p>