Autologous Transplantation Of Endothelial Progenitor Cells To Prevent And Treat Multiple Organs Dysfunction In Porcine

Trauma can cause severe damage of the target cells.Infectious sepsis and noninfectious systemic inflammatory response syndrome(SIRS) can interact eachother which will cause the dysfunction of self-repairing.What is more,the injury of blood capillary and microcirculation disturbance caused by dysfunction of self-repairing can result in multiple organ dysfunctions, which is deemed to be the beginning of the multiple organ dysfunction syndromes(MODS). Nowadays MODS turn to be the most frequent cause of death in patients admitted to intensive care units.Recent research shows that EPC is one kind of progenitor cells from bone marrow which can be mobilized by physio-stimulation and patho-stimulation to peripheral blood to differentiate into mature endothelial cells(EC) to encourage angiogenesis and differentiate to different kind of cells to process repairing.EPCs were originally thought to be present only during embryonic development.However,accumulating evidence in the past several years suggested that EPCs were the major progenitor cells to participate inphysio-angiogenesis and patho-angiogenesis in adult lives.EPCs could particate in reparing of dysfunctions of heart,renal,liver and pulmonary and the EPCs which metastasized to pars affecta could be reacted to release the vasoactive substance,growth factors,immunoregulatory cytokines and chemotatic factors.Two different EPCs subpopulations have been described,denoted as early and late EPCs with distinct cell markers.Early EPCs come from bone marrow or be mobilized to peripheral blood shortly,they all express progenitor-specific markers,including CD133(AC133),CD34 and KDR;but they don't express VE-cadherin and vWF.Late EPCs are the early cells which mobilized into peripheral blood or cultured in vitro;they can express the endothelial-specific markers,including CD31 and KDR instead of CD133 and CD34.The late EPC can take up acetylated LDL and UEA-1.CD133 and KDR which are deemed to be the key markers to identify the EPCs are the cell markers of haemopoietic stem cells and ECs and they can also express in embryo-vessels.The method of isolation and culture in vitro of EPC had not come to an agreement in recently studies.The method in this research was based on the summary of the national and international studies.EPCs were isolated from bone marrow monocular cells(BMMC) by density gradient centrifugation,and were cultured in vitro by some specific growth factors such as VEGF and so on.Then EPCs were identified by cell morphology,phenotype,cell proliferation,function of angiopoiesis and taking up acetylated LDL and UEA-1. Our research shows that EPC can metastasize to pars affecta for neovascularization of ischemic or injured tissue and amelioration of functional impairment caused by trauma.So autologous transplantation of EPC could step down the incidence of MODS and improve the prognosis of MODS.This research investigated the pathogenesis of MODS from the aspects of EPC repairing function,and the theory of autologous transplantation of EPC to prevent and treat MODS in porcine.This study was divided into four parts.The first part of our study was to replicate a porcine model of MODS,and then we monitored the number and function of EPC in peripheral blood in the stages of MODS.We used the standard method of isolation,culture and identification of EPC to proliferate EPC in vitro,and then did autologous transplantation of EPC to prevent and treat MODS.Part 1 Establishment a porcine model of MODSThe first part of our study was to replicate a porcine model of MODS which was characterized by the development of delayed two-phase process and it was the foundation of our investigation of autologous transplantation of EPC to prevent and treat MODS.Nineteen healthy male minipigs weighing 22～30Kg were randomly divided into two groups.One group was subjected to hemorrhagic shock plus endotoxiemia(group M,n=10).Another group was normal control only with anesthesia and sham operation(group C,n=9).Blood specimens were collected every 24 hours during the seven-day observation for the detection of serum GPT,GOT,Cr,BUN and arterial blood gas analysis,which were used to judge if MODS occured by compared with the initial value of itself.Histological changes of the main organs were observed under light microscope(LM).The mobidity and mortality of MODS in group M were 88.9%and 77.8%respectively,both much higher than group C.The two-hit model of MODS was a successful animal model which conform to clinical course, also with high mobidity and mortality.And the model was easy to duplicate.Part 2 The change of number and function of EPC in peripheral blood in steps of MODSIn the second part,we monitored the number and function of EPC in peripheral blood in the various stages of MODS to supply the theory of autologous transplantation of EPC to prevent and treat MODS.Ten microliters of whole blood which were got at different stages(normal,postoperation,2 hours after hemorrhagic shock,2 hours after blood retransfusion,1 hour, 12 hours,24 hours,48 hours,96 hours after endotoxin transfusion) treated by density gradient centrifugation to isolate the PMC;and then incubated the PMC for 30 min in the dark with PE-labeled monoclonal antibody KDR and the FITC-labeled monoclonal antibody CD 133. The cells which were double positive were deemed as EPC in peripheral blood.The number of double positive cells was counted by flow cytometry.The PMC were processed the function detection including:proliferation,adherence,metabasis and angiopoiesis after been culture for 96 hours.The number of EPC in peripheral blood at various MODS stages was reduced sharply and the functions of EPCs were also impaired than normal.Part 3 Standardization of Isolation,Culture,Identification and Function of Endothelial Progenitor Cells from Porcine Bone MarrowWe investigate to get the standard method of isolation,culture and identification of endothelial progenitor cell from porcine bone marrow for EPC transplantation.BMMCs were isolated by the method of density gradient centrifugation from bone marrow and were cultured by 1×10~6/cm~2 original density with specific culture solution for EPC.After 27 days of culture,the P6-EPC were identificated by taking up Dil-ac-LDL and FITC-UEA-1,flow cytometry testing,immunohistochemistry testing,ultrastructural organization testing and functoin of angiogenesis testing.The attaching cells would appear after 48h culture,and the cells would be clustering after 6 days culture.The Weibel-Palade body would appear in EPCs.And more than 85%EPC could take up Dil-Ac-LDL and FITC-UEA-1.CD133(+),CD34(+),CD31(++),KDR (++) would appear in the EPC by immunohistochemistry testing and the positivity 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.EPC could be positive by angiogenesis testing.Part Four Autologous transplantation of endothelial progenitor cells to prevent and treat MODSThis section aimed to investigate the effect of autologous transplantation EPC from bone marrow for post-traumatic multiple organ dysfunction treatment,and compare effect of treatment with different dose of EPC transplantation.Experimental animal bone marrow was taken in advance in accordance with the aforementioned method of EPC isolation,culture and amplification.The MODS animals were randomly divided into three groups,in accordance with the 1×10~6 cells/Kg body weight (low-dose transplant group,LT group,9),and 1×10~7 cells/Kg body weight(High-dose transplantation group,HT group,9) autologous transplantation in the treatment of EPC,and the group with no transplantation as the control group(M group,10).The results showed that:the mobidity and mortality of the experimental animals of LT group transplantation(75%;6/8,75%;6/8) was significantly higher than those of HT transplantation group(50%;4/8,37.5%;3/8),but lower than those of MODS group(90%; 9/10,80%;8/10)(P＜0.01).And the survival time of experimental animal of LT group(78.47±44.12 hours) was significantly shorer than that of the HT group(156.18±72.87 hours).And the WBC,GRAN,SALT,SAST,Cr,BUN of LT group was much worse than HT group,but slightly better than the control group.The results would suggest that autologous endothelial progenitor cell transplantation could improve the post-traumatic rehabilitation and reduce the mobidity and mortality of MODS.Conclusion:The reduction and dysfunction of EPC may be the key to the development of MODS,and autologous transplantation of endothelial progenitor cells in vivo can migrate to different organizations and repaire the lesions.Autologous transplantation of endothelial progenitor cells can improve the function of organs with post-traumatic ischemia and hypoxia. So it can step down the mobidity and mortality of MODS and prevent the development of the MODS.