Variation And Significance Of Endothelial Progenitor Cells In Mini-pigs With Multiple Organ Dysfunction Syndrome Caused By Trauma

In 1997,Asahara separated a subpopulation of progenitor cells called endothelial progenitor cells from the peripheral blood mononuclear cells at first.The endothelial progenitor cells have the ability of proliferation,migration,differentiate to adult endothelial cells.The multiple organ dysfunction syndromes(MODS) is the most common cause of death in the clinical dangerous patients.The pathogenesis of MODS is not clear and there are not satisfactory therapic methods in clinic at present.With the development of clinical technologies of progenitor cells,the moment emerged in pathogenesis reaserchs and clinical therapy of MODS.Some studies indicate that the progenitor cells have the ability to proliferate,migrate,differentiate and repair damaged organs with guiding of some cytokine.The endothelial cells are not only the damaged target cells,but also causes of blood capillary damage and dysfunction or the first component element of MODS. The massive animal and clinical experiments have already certificated that when tissues is damaged,especially ischemia,the proliferation,migration,differentiation of EPCs are reinforced into peripheral blood and turned into endothelial cells and replaced the damaged endothelial cells in tissue.The denuded damaged vessel is repaired by new endothelial cells.EPCs participate neovascularity in ischemia or damaged tissue and improve ischemia organs' function.If the proliferation,migration,differentiation of EPCs are seriously disturbanced after trauma,they would make the microcirculation unrepaired and MODS come bad to worse.To reaserch a standard method of isolation,culture and identification of endothelial progenitor cell derived mini-pig bone marrow.At the basement of animal model of MODS by "two-hit" injury,the number of endothelial progenitor cells in bone marrow and peripheral blood were determined with flow cytometry.We observe dynamic variation of bone marrow and peripheral blood endothelial progenitor cells in the various stages MODS caused by trauma,and investigate variant regularity for bone marrow endothelial progenitor cells during MODS caused by trauma.The studies will be illustrated by three parts as follows.PartⅠObjective:Objective:To set up a suitable method of isolation,culture, regeneration and identification of endothelial progenitor cells derived from porcine bone marrow.Method:We used density gradient centrifugation to isolate BMMC from bone marrow and culture BMMC by 1×10~6/cm2 original density with specific culture solution for EPCs.After 27 days of culture,the P6-EPCs by taking up Dil-ac-LDL and FITC-UEA-1,flow cytometry testing,immunohistochemistry testing,ultramicrostructural organization testing and function of angiogenesis testing were identificated.We found that the attaching cells would appear after 48h culture,and the cells would be clustering after 6 days culture.The Weibel-Palade body would be seen in EPCs.And we found more than 85%EPCs could take up Dil-Ac-LDL and FITC-UEA-1.CD133(+),CD34(+), CD31(++),KDR(++) would be seen in the EPCs by immunohistochemistry testing and the positive rate of CD133 would be 18.23±7.12%;the positivity of would be 47.71±14.85%;the positivity of CD31 would be 71.61±13.51%;he positivity of KDR would be:87.24±11.40%by flow cytometry testing.EPCs could be positive at angiogenesis testing.Density gradient centrifugation to isolate can be used to culture EPCs. Conclusion:The EPCs cultured in vitro can amplified and go down to future generation. BMMC from bone marrow can differentiate to EPCs under special culture situation. Anchoring factor and vascular endothelial growth factor are essential to culture EPCs in vitro.PartⅡObjective:The 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=9) 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 by iv administration of lipopolysaccharide(sigma,1mg/kg) within 24 hours.The function of heart,lung,kidney,liver and stomach intestine was 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 pathology.Compared with control group,with significant difference in incidence of dysfunction of lung(80.0%),gastrointestinal tract (70.0%),liver(50.0%),kidney(30.0%),heart dysfunction(20.0%),The morbidity and mortality of MODS in group M was 90.0%and 80.0%respectively,higher significantly compared with group C.Conclusion:This "Two-hit" way can make mini-pig MODS model successfully.Incidence rate and mortality of MODS is high.It is easy to reproduct.The models are resemble to typical clinical two-phase MODS.PartⅢObjective:To observe variation of bone marrow and peripheral blood endothelial progenitor cells in the various stages MODS caused by trauma,and found out variant regularity of bone marrow endothelial progenitor cells during MODS caused by trauma.Method:MODS group(n=9),control group(n=9).Bone marrow and peripheral blood were sampled at five time points(T1,T2,T3,T4,T5) during whole animal experiment.Of which 1ml were added into erythrocytic lysate,and it were calculated by "four-grid" method,then number of leucocytes in every sample was calculated.The rest sample and first antibody and second antibody for CD133 and KDR were added into flow tube.Number of endothelial progenitor cells in bone marrow and peripheral blood were determined with flow cytometry.The number of EPCs was obtained by number of leukocyte multiplying percentage acquired by flow cytometry,and at last the results were analyzed statistically.Results:The number of EPCs in normal bone marrow and peripheral blood was 7.64±0.68×10~6/L,3.54±0.26×10~6/L respectively.The number of leucocyte in marrow and peripheral blood elevated significantly,then descending markedly(P＜0.05),but amplitude of leukocyte elevating in marrow was higher than that in bone marrow significantly post-trauma(P＜0.05).Content of EPCs in peripheral blood elevated markedly at early stage of trauma(P＜0.01),then ascended obviously(P＜0.05),and kept descending especially as MODS developing.Moreover,the content of EPCs in blood descended obviously following pathogenetic condition became aggravated.The number of EPCs in bone marrow descended at short time firstly,then elevated obviouly,and persisted at high level.They descended as developing of MODS.Conclusion:The number of EPCs in normal bone marrow is more than in peripheral blood.The number of endothelial progenitor cells in peripheral blood elevates sharply at earlier period,then descended quickly.The number of EPCs in normal bone marrow descended wildly at first, then elevates obviously and persisted at high level during MODS caused by trauma in mini-pig.With the aggravating of MODS,the depression of EPCs in bone marrow emerges.Content of EPCs in bone marrow does not change markedly.Conclusion:Density gradient centrifugation can be used to isolate EPCs.The EPCs cultured in vitro can amplified and go down to future generation.BMMC from bone marrow can differentiate to EPCs under special culture situation.Anchoring factor and vascular endothelial growth factor are essential to culture EPCs in vitro.The "Two-hit" way can be used to make mini-pig MODS model successfully.It is easy to reproducd. The models are resemble to typical clinical two-phase MODS.we observed dynamic variation of bone marrow and peripheral blood endothelial progenitor cells in the various stages MODS caused by trauma,and found out variant regularity of bone marrow endothelial progenitor cells during MODS caused by trauma.The number of endothelial progenitor cells in peripheral blood elevates sharply at earlier period,then descended quickly.The number of EPCs in normal bone marrow descended wildly at first,then elevates obviously and persisted at high level during MODS caused by trauma in mini-pig. With the aggravating of MODS,the depression of EPCs in bone marrow emerges.