Differential Proteomics Analysis Of Polymorphonuclear Neutrophils Trans-endothelial Migration And Stagnation In Burn Blister Fluid In Vitro

OBJECTIVE To observe the proteomics changes of polymorphorphonuclear neutrophils(PMN) trans-endothelial migration and stagnation in burn blister fluid in vitro,PMN trans-endothelial migration and stagnation model were built and proteomics analysis was used to identify the biomarkers of inflammation after burn injury and explore the pathogenesis of burn sepsis.METHODS Normal human PMN and serum were harvested from healthy volunteers,burn serum isolated from massive burn patients within 48 hrs after burn injury,and burn blister fluid was collected from intact blisters on burn wound within 24 post burn hours(PBH).PMN was isolated from peripheral blood by dextran sedimentation and Ficoll sedimentation.PMN transmigration followed adhesive via HUVEC stimulated with burn serum and stagnation in burn blister fluid model were established using transwells^TM inserts.The total proteins of PMN in healthy volunteers(A group),transmigration followed adhesive(B group),stagnation in burn blister fluid(C group) were separated by immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis(2-DE),and the 2-DE images were analyzed by PDQuest 7.1.0 software.Discrepant expression protein spots were analyzed by Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) and peptide mass fingerprinting(PMF) obtained and searched in database to identify of the proteins.RESULTS 33 differentially expressed proteins spot were found between normal PMN group and the others groups,and 22 spots(16 proteins) were identified,including:Profilin-1,Actin,Heat shock 70 kDa protein,Peptidyl-prolyl cis-trans isomerase A,Leukocyte elastase inhibitor,Fructose-bisphosphate aldolase A,Catalase,Alpha-enolase, Vimentin,Tubulin beta chain,Lactotransferrin,Chloride intracellular channel protein 1,Glyceraldehyde-3-phosphate dehydrogenase,Protein DJ-1,Protein S100-A9 and F-actin-capping protein subunit alpha-1.CONCLUSIONSâ‘ PMN trans-endothelial migration model was established using transwells^TM inserts and a new stagnation model of PMN co-culturing with burn blister fluid model was built.â‘¡Well-resolved,reproducible 2-DE patterns of PMN from healthy volunteers,transmigration followed adhesive and stagnation in burn blister fluid were established.â‘¢Significant differences were found in the proteomics of PMN trans-endothelial migration and stagnation in burn blister fluid compared with normal PMN.Some proteins involve in the inflammation amplification and immune disorder and PMN apoptosis delay,and the others play an important role in capillary permeability.