Study On Transplantation Of Endothelial Progenitor Cells Transfected With Lentiviral Vector Carrying The Double Expression Genes Of HVEGF₁₆₅-GFP To Prevent And Treat MODS In Rabbits

Multiple organ dysfunction syndrome (MODS) has been the most frequent cause of death in patients admitted to intensive care units. In recent years, most of researchers have made a common agreement that one of the most important mechanisms of MODS is the unbalance between the injuries and repair to systemic capillary vascular endothelium, when the body gets severe injury.Since Ashara firstly isolated the endothelial progenitor cells (EPCs) from peripheral mononuclear cells in 1997, researchers had paid more and more attention on EPCs because these cells have a pivotal potency that they could differentiate into matured endothelial cells and then repair the endothelium. Most of the EPCs circulating in the peripheral blood come from bone marrow in the stimulation of all kinds of physiogenic or pathological factors. Numerous experiments in vitro or in vivo have demonstrated that, in the physiogenic or pathological conditions, EPCs are the most important cells that can repair vascular endothelium and promote the angiogenesis of organs. Furthermore the injuries of microcircu-lation in the single organ such as heart, liver, lung and kidney could be repaired by EPCs too.Vascular endothelial growth factor (VEGF)is a special mitogen for vascular endothelium. It can maintain the homeostasis of endothelial cells, induce the vascularization, enhance vascular permeability and maintain normal function of vessels. It is deem to the most superactive factor that can promote vascularization happen and make EPCs defferentiate into endothelial cells (ECs). Since the carrier system of lentivital vector (LV) has high and stable effectiveness for gene transfer and low adverse effect, it has been used more and more comprehensively in transgenes therapy for all kinds of diseases. In addition, the green fluorescent protein (GFP),which can express the fluorescent signal, has been used to observe gene expression and site-specific of transplantation cells in vivo. The discovery of GFP makes the development and variation of biological tracing come true in molecular and cellular level. In our research, we used the lentivral vectors to transfect the double express gene-hVEGF165-GFP with EPCs. Secondly, we studied the function of post-transfected EPCs in proliferation, immigration, differentiation and antiapoptotic ability for inflammatory environment. At length, we transplanted either untransfected or post-transfected EPCs into the MODS animal in order to figure out their distribution in vivo and observe the preventive and therapeutic effect of MODS.There were three parts in our study. In the first part, we constructed and optimized the system for the isolation,cultivation and identification of EPCs in order to proliferate EPCs fastly in vitro. In the second part, we constructed the carrier system of LV-hVEGF165-GFP to transfect with EPCs and identified the function of post-transfected EPCs in proliferation, immigration, differentiation and anti-apoptosis ability to inflammatory environment. In the third part, we transplanted the untransplanted or post-transplanted EPCs into animal model respectively to observe these cells'distribution in vivo and then investigated the mobidity and mortality of MODS animals. In the last part, we would assess the value of EPCs' transplantation and make an approach to the putative mechanism of MODS.Part 1 Isolation, Cultivation, Identification of endothelial progenitor cells from rabbit bone marrow in vitroObjective:to optimize the system for isolation, cultivation and identification of endothelial progenitor cells from rabbit bone marrow for transplantation.Methods:BMMCs were isolated by the method of density gradient centrifugation from bone marrow and cultured as 1×106/cm2 original density with specific culture medium for EPCs. After14 days of cultivation, the P3-EPCs were identificated by taking up Dil-ac-LDL and FITC-UEA-1,flow cytometry testing,immunohistochemistry testing,ultrastructural organization testing and the functoin of angiogenesis testing.Result:The attaching cells appeared after 48h culture, and these cells got clustering after 6 days culture. The Weibel-Palade body, which is the special characteristics for EPCs, appeared in our cultured cells. And more than 80% EPCs could take up Dil-Ac-LDL and FITC-UEA-1. CD133 (+),CD34 (+),CD31 (++),KDR (++) appeared in these cells by immunohistochemistry testing. Our cultured cells also were positive in angiogenesis testing.Conclusion:Density gradient centrifugation is a stable method to isolate BMMCs from the peripheral blood. It could provide very pure EPCs by this specific cell-culture way in vitro. Further more, these EPCs have all the normal function that they should have!Part 2:Construct a lentiviral vectors that carry both hVEGF165 and GFP Gene, Identify the function of those post-transfected EPCsObjective:to construct the lentivital vectors that can stably carry the double express gene of hVEGF165-GFP and can transfect with EPCs high effectively. To improve the EPCs' ability of proliferation,migration,differentiation, angiogenesis and the resistance for ischemia,anoxia,inflammatory factors.Methods:Firstly, we designed the hVEGF165 PCR primers:hVEGF165-F:5'-CGG GAT CCA TGA ACT TTC TGC TG-3'; hVEGF165-R:5'-CGA CGC GTC CGC CTC GGC TTG TCT-3'. Secondly, the plasmid of pDC316-hVEGF165 was used to be the template to amplify hVEGF165 by PCR. Combination and transformation would be performed when both the products of PCR and the plasmid of pWPXL-MOD had been cut by the enzyme of BamH I,Mlu I. This new acquired plasmid of pWPXL-MOD, which contains the exogenous gene of hVEGF165-GFP, was spreaded on the LB plate and incubated at 37℃overnight. As soon as the monoclones appeared on the plate 12-16 hours later, they would be picked up and expanded to extract the plasmids DNA. Thirdly, these acquired plasmids DNA were cut by the restriction enzyme of BamH I,Mlu I, and then the fragments of these recombinant plasmids DNA were identifed by electrophoresis. When the size of fragment had been confirmed, the sequence of targeting gene would be checked to make asure that it was definitely correct.We used the four plasmids system to package the lentivrial vector, including pRsv-REV, pMDlg-pRRE, pMD2G and the recombinant plasmid of pWPXL-MOD which contained the exogeneous gene of hVEGF165-GFP. The three orther plasmids of pRsv-REV,pMDlg-pRRE and pMD2G had all the imperative components for viral packaging. After these four plasmids had been prepared, they would be used to transfect with 293T cells in the protocol of calcium phosphate precipitation.8 hours after transfection, the current medium must be changed to the maximal medium and these 293T cells should be continued to culture for 48-72 hours untill the viral vectors were secreted from them. The supernatant, which contained abundant lentiviral vectors, was collected to concentrate to high concentrated solution. In the last step, the virus titer was measured by means of coubling dilution.The pure frozen EPCs were thawed and planted into the culture dish in the density of 1×105/cm2. As soon as the EPCs had become 60% confluence on the bottom of dish, the culture medium should be changed. At the same time, the concentrated lentiviral vectors were diluted to transfect with EPCs in the MOI for 50. These EPCs were co-cultured with lentiviral vectors for 24h and would be passaged as soon as they were 80%-85% confluence. These post-transfected EPCs could be amplified to a large number after several passages. At last step, we identified the function of these acquired EPCs in proliferation,migration,differentiation, angiogenesis and the resistance for ischemia,anoxia,inflammatory factors.Result:The final titer of lentiviral vectors was 5.0×108 TU/m in the concentrated viral solution. For these viral vectors, the average of efficiency for transfection was 87.9%±5.6%, when EPCs were transfected in the MOI for 50. The function of post-transfected EPCs showed that the ability of migration and angiogenesis were a little bit more enhanced, however, the proliferation,differentiation and the anti-apoptotic ability were significantly enhanced.Conclusion:When EPCs were transfected by our four plasmids system lentiviral vectors, the gene of hVEGF165-GFP carried by viral vectors could be transfered into these cells' genome. Furthermore this exogenous hVEGF165-GFP gene could enhance the function of EPCs in proliferation,migration,differentiation,angiogenesis and anti-apoptotic ability. These carriers of lentiviral vectors could play a critical role in the acquirement of high anti-apoptotic and more powerfully targeted migratory EPCs. However, the improvement for the ability of post-transfected EPCs in differentiation made these kinds of projenitor cells transfer to mature cells faster than untransfected ones. This characteristic could reduce the self-renewal potentiality of EPCs in vitro.Part3:Transplantation of untransfected and post-transfected endothelial progenitor cells to prevent and treat MODSObjective:to investigate the effect of transplantion of the post-transfected EPCs that come from bone marrow and assess their clinical value on post-trauma multiple organ dysfunction syndrome treatment. Moreover, we aim to compare the effect of treatment in the transplantation with the untransfected and post-transfected EPCs.Methods:Experimental animal bone marrow was in advance taken in accordance with the aforementioned method for EPCs isolation, culturation and amplification. The MODS animals were randomly divided into five groups:A:transplantation with LV/ hVEGF165-GFP-EPCs to the MODS animals (12 rabbits); B:transplantation with LV-GFP-EPCs to the MODS animals (12 rabbits); C:transplantation with untransfected EPCs to the MODS animals (12 rabbits); D:intramuscular injections of hVEGF165 to the MODS animals (12 rabbits); E:none treatment for MODS animals as the control group (12 rabbits). The distribution of transplanted EPCs in main organs was observed in the use of fluorescence microscope and the improvement of these main organs was also assessed after transplantation.Result:The mortality of the experimental animals for untransfected EPCs transplantation group (50%; 6/12) was significantly higher than those of LV/hVEGF165-GFP-EPCs transplantation group (25%; 6/12), but lower than those of injection of hVEGF165 group (83.3%,10/12) and MODS group (75%; 9/12) (P＜0.05), however, there was no statistical difference to LV-GFP-EPCs group(58.3%; 7/12) (P＜0.05). In addition, the survival time of experimental animal in LV/hVEGF165-GFP-EPCs group (136.48±42.27hours) was significantly longer than those in LV-GFP-EPCs group (87.79±24.12 hours), untransfected EPCs group (90.34±23.92 hours), injecting hVEGF165 group(59.21±20.35 hours) and MODS group(63.45±23.55 hours). Moreover, the examine result of WBC, GRAN, SALT, SAST, Cr, BUN in LV-GFP-EPCs group had no difference to those in untransfected EPCs group, but was slightly worse than those in LV-VEGF-GFP-EPCs group, and significantly better than those in the control group.Conclusion:In our study, we proved that exogenous hVEGF165 gene could significantly improve the anti-apoptotic ability of the EPCs when they had been transferred into EPCs gemone. Furthermore, these post-transfecetd EPCs definitely could enhance the repair ability for microcirculation. In short, EPCs transplantation could improve the post-traumatic rehabilitation and reduce the mobidity and mortality of MODS animals.Summary:When the body suffers to severe inflammatory trauma, the reduction and dysfunction of EPCs may be the key point in the development of MODS. Since the transplanted EPCs could migrate to different organs or tissue and transfer to vascular ECs in vivo, these EPCs might play a critical role to repair the microcirculation and could enhance the ability of angiogenesis, both of which can ameliorate the organs'dysfunction. When EPCs were transfected by the lentiviral vectors that carried hVEGF165-GFP gene, they could become stronger in the anti-apoptotic ability for SIRS and possess higher target therapeutical effect on the injured organs and circulation. These transplanted EPCs could prevent MODS, improve the MODS animal's prognosis and extend their survival time.