Study On The Change Of Apoptosis In Rabbits Of Multiple Organ Dysfunction By Transplanting Of Endothelial Progenitor Cells Transfected With Lentiviral Vector Carrying HVEGF165-GFP Gene

Multiple organ dysfunction syndrome (MODS) is the most frequent cause of death in patients admitted to intensive care units. Severe sepsis and septic shock are the primary causes of MODS and develop as a result of the host response to infection of Gram-neagitve and Gram-positive bacteria. Infections sepsis and nonsepsis systemic inflammatory response syndrome (SIRS) encompass a complex mosaic of interconnected events. 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 MODSRecent 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.Also research finds that 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 turn into endothelial cells and replace the damaged endothelial cells in tissue. The denuded damaged vessel is repaired by new endothelial cells. EPCs can participate neovascularity in ischemia or damaged tissue and improve ischemia organs' function. If the proliferation, migration and differentiation of EPCs are seriously disturbanced after trauma, they would make the microcirculation unrepaired and MODS come bad to worse.Past studies showed that the cytokines related to MODS were concluded IL-1, IL-6, TNF, HSP, Glucocorticoid, LPS, and so on. They were associated with apoptosis. So we may be more in-depth understanding of the mechanism of MODS by studying the mechanism and influencing factors of apoptosis and provide the basis for clinical.The study was to replicate a rabbit model of MODS, and then did transplantation of EPCs transfected with LV/VEGF165-GFP gene. Then we observed vital signs of major organs and the expression of inflammatory cytokines, such as IL-1β, TNF-αand so on. Our purpose was to find the effect of the EPCs to prevent and treat MODS. The study includes three main parts.Part 1 Objective:To replicate a rabbit model of MODS which was characterized by the development of delayed two-phase process and it was the foundation of our investigation of transplantation of EPCs to prevent and treat MODS. Methods: Twenty-four healthy male rabbits weighing 2.25～2.79Kg were divided into two groups randomly. One group was subjected to hemorrhagic shock plus endotoxiemia (group M, n=12). Another group was normal control only with anesthesia and sham operation (group C, n=12). 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). Results:The mobidity and mortality of MODS in group M were 83.3% and 75.0% respectively, both much higher than group C. Conclusion:The two-hit model of MODS was a successful animal model which conforms to clinical course, also with high mobidity and mortality. And the model was easy to duplicate.Part 2 Objective:To construct the Lentivirus carrying hVEGF165-GFP gene, and transfect endothelial progenitor cells in vitro. Methods:The VEGF165 gene was ligated into the lentiviral overexpression vector pWPXL-MOD. Then we used lentiviral packaging plasmid to transfect 293T cells and got the LV/hVEGF165-GFP. The LV/hVEGF165-GFP was co-cultured with EPCs. Adhesion, migration, proliferation and angiogenesis function were observed after tranfected. Results:Lentiviral vector expressing the target gene VEGF was successfully constructed. It confirmed that the LV/hVEGF165-GFP's transfection rate was nearly 99%. After transfected, EPCs' migration, proliferation and angiogenesis were increased (P<0.05). Conclusion:LV/hVEGF165-GFP was non-toxic to EPCs, and can improve the EPCs' adhesion, migration and proliferation ability.Part 3 Objective:To study the change of apoptosis in rabbits of multiple organ dysfunction syndrome by transplanting of endothelial progenitor cells transfected with LV/VEGF165-GFP gene. Methods:Thirty-six healthy male rabbits weighing 2.25～ 2.79Kg were divided into three groups randomly. One group was subjected to hemorrhagic shock plus endotoxiemia (group M, n=12), the group with no transplantation as the control group. Another group (group ET, n=12) was transplanted of EPCs with the 1×107 cells/ Kg body weight. The other group (group VT, n=12) was transplanted of EPCs transfected with LV-VEGF165-GFP gene with the 1 x 107 cells/Kg body weight. 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. Histological changes of the main organs were observed under light microscope. The apotosis of the main organs was tested by TUNEL. The caspase-3 mRNA of the main organs was measured by RT-PCR. Results: The WBC, GRAN, GOT, GPT, Cr, BUN and other indcators of VT group were significant improvement compared with ET group and M group. The apotosis and caspase3mRNA expression of main organs in VT group were lower than ET group and M group (P<0.01). The mobidity and mortality of VT group were lower than ET group and M group (P< 0.05). Conclusion:Transplantation of EPCs transfected with lentivirus-mediated VEGF165-GFP gene can reduce the MODS animal apoptosis, make a marked improvement of the function in main organs and reduce the animal's mortality.