Study Of The Change Of Organ's Microvessel Density In The Treatment Of MODS After Transplantation Of Endothelial Progenitor Cells Carrying The Gene Of HVEGF165-GFP Transfected With Lentiviral Vector

Multiple organ dysfunction syndrome (MODS) is one of the most common causes of death in the clinical treatment for severe cases. The newest research shows that, event it is unclear for its true mechanism of pathogenesis, systemic inflammatory response syndrome (SIRS) would make the auto-repair of body disorder, and induce the capillary vascular injury and microcirculation disturbance. This may be not only the cause of MODS but also the first step of MODS.Endothelial progenitor cell (EPC) is a kind of stem cell that is the precursor cell of the matured endothelial cells. These cells both could unceasingly proliferate themselves and induced differentiate to matured endothelial cells. EPC would not only take part in the embryonic angiogenesis but also play a great role in the postnatal vasculogenesis and the repair process for the injured vascular endothelium. It is one of hotspots in the research to repair injured vascular endothelium.A lot of animal and clinical experiments have showed that, when the body gets hurt, EPC could differentiate into matured endothelial cell to take the place of the handicapped endothelial cells and repair the area of injured vascular endothelium. Further more, it also takes part in the process of neoangiogenesis for the ischemic or injured area and then improves the ischemic condition of organs. However, if the body was excessively injured, the differentiation,migration and proliferation of EPC would turn to disorder severely, and induce the local microcirculation too badly hurt to repair that result in MODS.Our research shew that transplanting EPC containing VEGF to treat the ischemic diseases can improve the regeneration of blood vessels. The incidence rate of MODS would significantly decrease by transplanting these kinds of EPC and the prognosis of MODS would get better. For these reasons, we used the lentiviral vector carrying VEGF-GFP to transfect EPC and investigate the cell characteristics of these kinds of EPC. In a word, our research would pave a way for the prevention and treatment of MODS.This study was divided into three parts. The first part was to replicate a rabbit model of MODS.The second part was to transfect the VEGF-GFP gene into EPC by lentiviral vector. And then the last part was to transplant the transfected EPCs into the MODS animal model to investigate the incidence rate of MODS and the changing of microvessel density in the important organs. Make an assessment for the effect of treatment and putative mechanism of MODS.Part 1 Establishment of a rabbit model of MODSIn the first part, we replicated a rabbit model of MODS which was characterized by the development of delayed two-phase process and it was the foundation of our investigation to prevent and treat MODS.24 healthy male rabbits weighing2.52±0.27Kg were randomly divided into two groups. One group of rabbits were subjected to hemorrhagic shock plus endotoxiemia (group M, n=12). Another group of rabbits were normal control only with anesthesia and sham operation (group C, n=12). The change of main organs'function was observed carefully by the monitor and the pathological changes of the main organs were judged by light microscope (LM). The mobidity and mortality of MODS in group M were 83.3% and 75.0% respectively, both much higher than group C. The two-hit model of MODS was a successful animal model conforms to clinical course, also with high mobidity and mortality. And the model was easy to duplicate.Part 2:Construction of a lentiviral vector that can express both VEGF and GFP and Transfection of EPCs in vitroIn this part we constructed the lentivital vectors that could stably express both VEGF and GFP, and transfected EPC high effectively. And then made the identification of cell function for these acquired cells, it would be the foundation for the treatment of transplantation.The first step was design and synthesis of the VEGF PCR primers and the amplification. Combination and transformation was made after cutting these PCR products, they were inserted into the lentiviral vector--pWPXL-MODcontaining GFP gene. When the positive clones formation were ready, picked up and cut them with the restriction enzymes of BamH I,Mlu I, at last the sequence was identified by electrophoresis checkingWe use the expression plasmids--pWPXL-MOD containing VEGF-GFP gene; the package plasmids-- pRsv-REV and pMDlg-pRRE; the envelope plasmids--pMD2G to construct a four plasmids system of lentivrial vectors from the calcium phosphate precipitation. We could get high concentrated solution of LV-VEGF-GFP by concentrating these vectors. Co-culture these lentivirus and EPC in the MOI for 50, the EPC could be transfected very well. Identify the function of proliferation. The results shew 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. Further more the report of gene sequencing shew that the sequence of insertion element in the recombinant plasmids is accordant with the sequence of objective gene of VEGF. In a word, we constructed and packaged the lentiviral vetctors successfully, and had proved that the abilities, comparing to normal EPCs, of the post-transfected EPCs in proliferation were significantly enhanced.Part 3 Study of The Change of Organ's Microvessel Density in The Treatment of MODS after Transplantation of Endothelial Progenitor Cells Carrying the Gene of hVEGF165-GFP Transfected with Lentiviral VectorThis section aimed to investigate the effect and safety of transplantation EPC containing VEGF-GFP gene for post-traumatic multiple organ dysfunction treatment, and then to compare the effect of treatment with different teams of EPC transplantation.MODS animals' model were constructed and randomly divided into three groups,①transplantation with EPCs (ET group,12).②transplantation with LV-VEGF-GFP-EPCs (VT group,12). And the amount of transplantated EPCs was 1×107 cells/Kg.③the group that there is no EPC transplantation would be control group (M group,12).The results shew that the mobidity and mortality of the experimental animals of VT was significantly lower than those of ET and M groups (P<0.05). The microvessel density of vital organs was significantly higher than ET group and M group.The results would suggest that transplantation of EPC containing VEGF-GFP gene could improve the post-traumatic rehabilitation, extend the survivor time and reduce the mobidity and mortality of MODS.