Study On The Effections Of Microthromosis In Rabbit MODS Models Transplantated With Endothelial Progenitor Cells Transfected With ThVEGF165-GFP Gene

EPC(endothelial progenitor cell) is a kind of cells which resides in the peripheral blood, bone marrow and organs and could differentiate into mature endothelial cells, working as stem cells. When the tissue damages are caused by trauma, inflammation or hemorrhagic shock, EPC could be released from bone marrow and released into the peripheral blood. Induced by chemokines such as VEGF, SDF-1, EPC assemble in the injury area and repair vascular endothelial which could improve the local blood supply and make a repairment of organizations. In recent years, studies on cultivation and applications of EPC to treat ischemic diseases have become a focus in medical researches.When body suffers serious trauma, shock or infection, at least two system or organ dysfunctions could be induced which is described as multiple organ dysfunction syndromes (MODS). MODS is the most serious complications of many diseases and the final outcome. Trauma and infection are two important causes of MODS, both of which could activate inflammatory cells, inducing excessive inflammatory response. Endothelial cell is the first target of inflammatory mediators. The injury and dysfunction of endothelial cells could lead to amplification of inflammatory response and ruin steady-state of internal environment, resulting in the occurrence of MODS.In this study, EPCs were cultured in vitro and transfecetd with VEGF165-GFP gene by lentiviral vector. After that, the cells were transplanted into rabbits which suffered MODS induced by hemorrhagic shock and LPS. The changes of microcirculation were observed and cytokines including ANG-1 and MCP-1 were measured which may reveal the therapeutic effect and related mechanism of EPC in the treatment of MODSThe experiment included four parts:First, marrow-derived mononuclear cells were cultured in vitro; Then, lentiviral vectors were constructed and the VEGF-GFP gene was transfected into EPC. The functions of cell proliferation were observed; in the end, EPCs were allotransplanted into the rabbits suffering MODS which were induced by hemorrhagic shock and LPS. The changes in post-transplant function of organs were observed and micro thrombi were counted as well as the level f EPC related cytokines (MCP-1, ANG-1).PartⅠCulture and Identification of Marrow-derived Endothelial Progenitor CellsIn this part, Marrow-derived mononuclear cells were cultured and amplified in vitro, so were the identification of EPC functions.Mononuclear cells were got and purified by density gradient centrifugations, inoculated in petri dishes in density of 1×105/cm2 using a culture media containing a variety of growth-promoting factors. Generations, morphological features, ultra structure, surface antigens (CD133, CD31, and KDR) and phagocytic experiments (FITC-UEA-1, Dil-ac-LDL), angiogenic functions in vitro were detected and identified in the tert cell.The results showed that:fusiform adherent cells emergenced in 24-48 hours after being cultured. The generation reached peak level at 6-10 days and the colony formation could be observed. The Weibel-Palade body was shown by electron microscope; positive rate of CD133-rate was 4%, the positive rate of CD34, and KDR was above 90%; adherent cells could specially uptake Dil-ac-LDL and FITC-UEA-1, showing the characteristics of endothelial cells. Tert cells could establish capillary network structures in vitro.PartⅡConstruction of a lent viral vector and Transfection of VEGF-GFP Gene into EPC in vitroIn this part, hVEGF/GFP fusion gene lent viral vectors were made and transfected into endothelial progenitor cells in vitro. The experiments provided a cell source for the next step of study.The VEGF PCR primers were designed, synthesized and amplified. The combination and transformation were made after these PCR products were cut, then inserted into the lent viral vector--pWPXL-MOD, which contains GFP gene. The positive clones formation were picked and cut by the restriction enzymes of BamH I,Mlu I, and electrophoresis checking were done to confirm that the sequence was correct.The expression plasmids--pWPXL-MOD were used in the experiment, which contained VEGF-GFP gene as well as the package plasmids--pRsv-REV, pMDlg-pRRE, the envelope plasmids-pMD2G. Then a four-plasmids system of lent viral vector were constructed. The LV-VEGF-GFP could be got in a high concentration when the vectors were concentred. Lent virus and EPC were co-cultured in the MOI for 50 and transfect ion could be accomplished very well. Then the functions of proliferation were identified. The results showed that when the objective gene of VEGF-GEP was inserted into the over-expression carrier of pWPXL-MOD, both the carrier fragment and objective fragment could express. Furthermore the report of gene sequencing showed that the sequence of insertion element in the recombinant plasmids was accordant to the sequence of objective gene of VEGF. In a word, we constructed and packaged the lent viral vectors successfully and proved that the ability of proliferation of post-transfected EPC is significantly enhanced contrast to the normal.PartⅢThe treatment of MODS by allotransplanting Endothelial Progenitors Cells transfected with Gene of ThVEGF165-GFPIn the parts, the MODS model was prepared and the EPCs were allogeneic transplanted as a treatment. Organ functions and micro-thrombosis in tissues were observed, as well as detection of EPC related cytokine (MCP-1, ANG-1).36 rabbits were randomly assigned into three groups and MODS model was constructed. The group without treatment was used as the control (M); group treated with MODS and transplantation of EPCs was the EPC treatment group (ET); the other 12 animals treated with MODS and transplatation of EPC transfected with hVEGF-GFP fusion gene was assigned as VT group. The point of transplantation was 1 hour after infusion of LPS and the dosage of cells was 1 x 107/kg weight. Organ functions were monitored. Animals were executed after 96h. Pathological examination and micro thrombus count were made. The ELISA method was used to detect levels of MCP-1 and ANG-1 in tissues.These results suggested that EPC transplantation effectively improved the functions of important organs, inhibited the micro thrombosis in circulation and reduce the mortality of MODS.