Polymorphonuclear Neutrophils Proteomic Study In Scald Staphylococcus Aureus Sepsis Rabbits

Objective To observe the distinct protein expression in circulatingpolymorphonuclear neutrophils(PMN) from scald sepsis rabbits infectedwith staphylococcus aureus and approach the pathophysiologymechanisms in PMN total cell protein level for beneficial clues in clinicaldiagnosis and therapy in scald(bum) sepsis.Methods rabbit models includes four groups:sham scald, scald,2 hours(hrs) after injection staphylococcus aureus(S.aureus) post-scald,6hrs after injection S.aureus post-scald,3 rabbits in every group.S. aureus(American Type Culture Collection, ATCC25923) in logarithmicphase be injected to aures vein of rabbits 24hrs after scald injury andblood be got through carotid artery cannula 2hrs,6hrs later respectively.Blood be got from Simple scald and sham scald groups through carotidartery cannula 24hrs after scald or sham scald treatment. Then PMN beisolated by gradient centrifugation and erythrocytes deposition andlysis. Proteomics techniques be employed to extract PMN totalprotein, immobilized pH gradient-based SDS-PAGE two-dimensional gelelectrophoresis be utilized to separate PMN protein, coomassie brilliantblue staining,Imagescanner scanning and PDQuest software be used todetect protein spots expressed differently among the experimentalgroups, spots incised from gels and digested by trypsin in-gel andmatrix-assisted laser desorption/ionization time of flight massspectrometry(MADI-TOF-MS) and Mascot software,MSDB,NCBIdatabase bank be applied to identify the possible scald sepsis specificproteins differently expressed.Results 1)the gel image maps have high resolution and goodreproducibility, among the 3 maps from the same group(sham scald group)PMN species, the protein spots expressed are 759±20, the average spotsmatching rate is 93%, the average deviations of matched protein spotsposition is 0.80±0.29mm in isoelectric focusing (IEF) direction and1.1±0.37mm in SDS—PAGE direction respectively.And maps in theother three groups have good resolution and good reproducibilitysimilarly.The matching rate between the average gel maps of every group is 81％～100％.2) 19 protein spots expressed differently are selected and theirpeptide mass fingerprints(PMF) are all clear,9 spots corresponding to 7proteins are identified by searching proteome database banks.Theproteins are: protein disulfide-isomerase precursor, actinβ, annexinⅠ,annexinⅡ, thiol-specific antioxidant,thioredoxin and hypothetical protein.protein disulfide-isomerase precursor and annexinⅠhave 2 isomersrespectively.3) annexinⅠand actinβare up-regulated in scald groups comparedwith sham scald group by image analysis, and the othersdown-regulated. The expression quantity of protein disulfide-isomeraseprecursor in scald group is 1/100 of the sham scald group,and thioredoxinexpression quantity approximate to zero in scald group.All the proteinsidentified do expressed differently between scald group and the twoscald sepsis groups but the differences are less prominent than thedifference between the scald group and the sham scald group.Conclusions 1) This study established well-resovled andreproducible immobilized pH gradient-based SDS-PAGE two-dimensional gel electrophoresis image maps of PMN from normal andscald sepsis rabbits,thus provide hepful resources for scald(burn) sepsisPMN 2-DE maps database.2) PMN proteins express differently in sham scald,scald,andscald sepsis rabbits, and the different expression of these proteins iscorrelated to impared functions of PMN in chemotaxis, phagocytosis andbacteriolysis, the PMN are detained in the microvascular and causedamage to the endothelial cells and tissue of organ, thus cause SIRS andsepsis. these proteins are potential molecular marks for early diagnosis forscald(bum) sepsis, and also give clues for new strategy in sepsis therapy.