Differential Proteomics Research Of DCs Treated With Heparan Sulfate---a New Vaccine Adjuvant

Objective:The subject mainly studies Heparan sulfate (HS) stimulate dendritic cells (DCs), which are the important antigen presenting cells (APCs) in the body, and then analysis the differential proteomics of DCs.Methods:(1) CD14+monocytes obtained from peripheral blood mononuclear cell (PBMC) with magnetic activated cell sorting (MACS), and cultured with rhGM-CSF and rhIL-4for5days to acquire the immature dendritic cells (imDCs). imDCs were divided into two groups according to whether treated by HS adjuvant and/or HBV antigen. They are A group:①blank control group;②adjuvant (HS) group and B group:①antigen (HBsAg) group;②antigen (HBsAg)+adjuvant (HS) group.(2) Extract the total proteins of DCs which were stimulated, and then a series of experiments had been performed to detect and analyse protein expression differences in DCs, such as protein precipitation and quantification, two-dimensional electrophoresis (2-DE), scanning, Image Master2D Platinum7.0analysis (Ratio>1.5), MALDI-TOF/TOF detection, and bio informatics analysis.(3) Relative expression differences of six key proteins were identified by Western Blot.Results:(1) In vitro, HS plays a little role in promoting DCs maturation, but probably plays a greater role in keeping immaturation of DCs to enhance those cells uptake and process antigen.(2)28differentially expressed proteins were found in A group (all of these proteins’expression are up-regulated), which were divided into7different function groups:Cytoskeleton proteins, Macromolecular Biosynthesis and Metabolism associate proteins, Organelle associate proteins, Molecular Chaperones, Cathepsins, Proteasome and Channel proteins.29differentially expressed proteins were found in B group (SERPINB1’s expression was down-regulated while others expression were all up-regulated), which were divided into5different function groups:Cytoskeleton proteins, Macromolecular Biosynthesis and Metabolism associate proteins, Signaling Transduction associate proteins, Cathepsins and Channel proteins. Functions of those57differentially expressed proteins were analyzed, and approved that most of these proteins are playing a role in immune response system. So the study provides a good research platform for researching the molecular mechanism of HS in cells.(3) CTSB, PSME1, LTA4H, LSP1, CTSD and SERPINB1were selected to identify the relative expression differences by Western Blot. Western Blot verified the validity of2-DE spots analysis by Image Master2D Platinum7.0and the6important proteins’relative expression differences. From our studies, we suggest that the6proteins play key roles in the adjuvant molecular mechanism of HS. They may play a role in uptaking and procesing antigen, promoting the maturation of DCs, inducing the cellular immune response.Conclusions:The study found the probably molecular mechanism of HS by differential proteomics, and provided many target molecule for adjuvant mechanism intensive study. We also can select several proteins to study intracellular protein interactions and subcellular localization when adjuvant stimulate DCs, then it can not only further improve the adjuvant mechanism research, but also can provide new targets for adjuvant development.