The Study Of Endothelial Progenitor Cells Modulated By P38mitogen-activated Protein Kinase In Multiple Organ Dysfunction Syndrome In Porcine

The multiple organ dysfunction syndrome(MODS) or multiple organ failuer(MOF) is the most common cause of death in the clinical dangerous population.At present the pathogenesis of MODS is not clear and there are not good thrapical methods in clinic.In 1997,Asahara separated progenitor cells called endothelial progenitor cells from the peripheral blood mononuclear cell first of all.The endothelial progenitor cells have the ability of proliferation,migration,differentiation and could turn into endothelial cells.With the development of clinical technologies of progenitor cells,the moment of MODS emerges in pathogenesis and clinical therapy.Some studies that indicate the progenitor cells have the ability of proliferation,migration,differentiation into damaged organs with VEGF in the peripheral blood and the differentiated endothelial cells can repaire damaged organs at the first place;The endothelial cells are not only the damaged target cells but also causes of blood capillary damage and dysfunction or the first ring-join of MODS.The massive animal and clinical experiments have already certificated that when tissues are damaged especially ischemia,the proliferation,migration,differentiation of EPC are reinforced in the peripheral blood and could turn into endothelial cells and replace the damaged endothelial cells in tissue,repaire denuded damaged endothelial cells in blood vessel,participate neovascularity in ischemia or damaged tissue and improve ischemia organs' function.As the serious disturbance of proliferation,migration,differentiation of EPC after trauma,they could make the microcirculation unrepaired in the damaged part and MODS will take place.This study is based on animal mode of MODS of "two-hit" injury and EPC's culture and identification in vitro,and all principal organs' metergasis,p38MAPK's phosphorylation,TNF-αmRNA and IL-1βmRNA's expression in the peripheral blood mononuclear cell,TNF-αand IL-1β's concentration and the quantity and function of the EPC with FCM are observed in the peripheral blood plasma in vivo;.and EPCs are cultured with TNF-αand IL-1β;the quantity and function of the EPC and p38MAPK's phosphorylation in EPC are monitored in vitro and the EPC were mediated by the TNF-αand IL-1βby the p38 mitogen activated protein kinase and the pathogenesis from cell dysdifferentiation are discussed in multiple organ dysfunction syndrome.The study is divided into 3 parts.PartⅠThe culture in vitro and bionomics of EPC.Methods:drew-off 20ml bone marrow in porcines and got mononuclear cells with density gradient centrifugation.The mononuclear cell suspension was inoculated in plate(diameter=10cm) with the density of 2×10~6/ml.Spindle-shaped cell populations after 2 days and adherent cobblestone-appearance cells and cytoreticulum or lumens' structures after 6 days were obsvered.The inoculated cells were propagated to P3.Theywere identificated with monoclonal antibody of CD133,CD34,CD31 and VEGFR-2 and they were positive.They were positive in double swallows with FITC-UEA-I and Dil-acLDL and they were positive in angiogenesis experiment.The adherent cells expressed VEGFR-2(87.24±11.40%), CD34(47.71±14.85%),CD133(18.23±7.12%) and CD31(71.61±13.51%).So they were the endothelial progenitor cells.After the endothelial progenitor cells of P3 inoculated with TNF-αand IL-1βstandard cytokine in vitro,the quantity,proliferation, migration,adherence and angiogenesis of EPC were observed.Result:the quantity and function of the EPC were dependent on TNF-αand IL-1βstandard cytokine concentration(P＜0.01) and they were correlated with p38MAPK's phosphorylation.The SB203580(1umol/L) that was p38MAPK's specific inhibitor could prevent the descent of the quantity and function.Conclusion:In vitro,TNF-αand IL-1βmake the quantity and function of the EPC descend through p38MAPK's phosphorylation,The SB203580 could prevent them.PartⅡThe porcine model of MODS with Wiggers' method that was characterized by the development of delayed two-phase process was firstly replicated.Method:Pigs were randomized into two groups:group C(n=10) as a control group,receiving anesthesia and sham operation only,group M(n=10) as a "Two-hit" injury model group,induced by hemorrhagic shock(50±5mmHg for 1.5-2hours) and resuscitation followed byⅳadministration of lipopolysaccharide(sigma,0.5mg/kg) within 24 hours.The function of hearts,lungs,kidneys,livers and gastrointestinal tracts were monitored successively and dynamically.The pigs were executed after 7 days and the principal organs' pathomorphology were observed.Blood specimens were collected every 24 hours during the seven-day observation for the detection of serum ALT,AST,Cr,BUN,CO and arterial blood gas analysis.Result:ALT,AST,Cr,BUN obviously ascended particularly before death(P＜0.01);CO,PaO2 obviously decreased particularly before death(P＜0.01). The idio-inflammation was the principal pathematology.Compared with control group,the significant difference in incidence of dysfunction of lung(80.0%),gastrointestinal tract (70.0%),liver(50.0%),kidney(30.0%),heart dysfunction(20.0%) were observed.The incidence of MODS and mortality in group M was 90.0%and 80.0%respectively,was higher significantly compared with group C.This "Two-hit" porcine model was characterized by the pathogenesis and manifestation analogous to clinical situation and fit for futher study.PartⅢThe p38MAPK's phosphorylation and TNF-αmRNA and IL-1βmRNA's transcription of the peripheral blood mononuclear cell and TNF-αand IL-1βand the quantity and function of EPC of the peripheral blood were observed in MODS.Method: In vivo,p38MAPK's phosphorylation with Western-blot,TNF-αmRNA and IL-1βmRNA with RT-PCR were monitored in the peripheral blood mononuclear cell,TNF-αand IL-1βwith ELISA and EPC with FCM were monitored in the peripheral blood plasma.Result:In vivo p38MAPK's phosphorylation of mononuclear cell was much more(P＜0.01),the synthesis and secretion of TNF-αand IL-1βrised in the peripheral blood mononuclear cell(P＜0.01).The quantity and function of EPC conspicuously decreased in the peripheral blood(P＜0.05).In vitro,the TNF-αand IL-1βcan make the quantity and function of the EPC descend through the p38MAPK's phosphorylation.Conclusions:phosphorylation is important to pathogenesis in MODS,the p38MAPK's phosphorylation makes TNF-αmRNA and IL-1βmRNA's transcription TNF-αand IL-1βof the peripheral blood mononuclear cell increased,The increased TNF-αand IL-1βof the peripheral blood plasma could make the quantity and function of EPC descend through p38MAPK's phosphorylation.So the inflammatory reation of MODS aggravated.Conclusion The p38MAPK's phosphorylation is important to pathogenesis in MODS, the p38MAPK's phosphorylation makes TNF-αmRNA and IL-1βmRNA's transcription of TNF-αand IL-1βof the peripheral blood mononuclear cell increase,The increased TNF-αand IL-1βof the peripheral blood plasma can make the quantity and function of EPC descend through p38MAPK's phosphorylation.