| Background liver transplantation is the best therapeutic regimen for Liver function failure in terminal stage. Both surgical technic and contemporary immunosuppressive drug treatment for transplanting have been developed greatly. The occurrence of organ loss by acute rejection has been decreased. But it is helpless for the chronic rejection. Organ transplantation will be state of final stage after operation, which was induce by vasculopathy of chronic rejection ,long-term using immunosuppressant leeding to toxic reaction and cardiovascular disease,then the parasitifer will be faced with retransplantation,even death. Therefore,the mechanism of immunotolerance has been studied for searching new approach of inducing immunotolerance,then the patients with transplantion could depart from risk of opportunistic infection and tumor occurred by long-term using immunosuppressant.It has been greatly developed in immunotolerance for many animal experiment and some clinical research., no rejection has been reported in some patients,which stopped using immunosuppressant all the year round. At present,the question to be faced with is how to induce stabilizing, lasting immunotolerance and determine formative immunotolerance. Recently, dendritic cell's critical role in liver transplantation immunotolerance has been thinked highly of gradually. Dendritic cells have capability of inducing form of immune tolerance and prolonging survival time of transplant. Research has indicateded that stimulating maturity of dendritic cell has been implemented by means of activation of nuclear factor kappa B, Among heterodimer, p50/Rel-A(p65)was major form to educe biological function. Developed technique of RNAi recently has provided original means to educe biological function, Effective gene therapy requires a reliable gene transfer tools to efficiently insert target genes into the cells of the grafts. Adenovirus vectors have been favored because of their propensity to infect hepatic cells and their ability to infect proliferation and nonproliferation cells.Part I: Generation of Rat Bone Marrow-derived DendriticCells in Vitro and Evaluation of Their Biological CharacterizationObjective To establish a suitable method for the generation of Lewis rat immature dendritic cells and evaluate their biological characterization.Methods Bone marrow cells were developed under the concentration of granulocytemacrophage colonystimulating factor , and the cell yield and the number of OX62-positive DC was determined at day 7. The phenotypic characterization were analyzed with flowcytometry,the capacity of stimulating T cells was determined by allogeneic mixed leukocyte reaction (MLR),and the levels of IL-12 secreted by DC and interferon-γ(IFN-γ) and IL-12 levels in 5-day mixed leukocyte culture supernatants was detected by ELISA.Results With 5% GM-CSF concentrations resulted in a higher number of cell yield and higher percentage of OX62 at day 7. Under the condition of GM-CSF(5ng/ml), DC at day 9 expressed an intermediate levels of MHCII and low levels of CD86, revealed a low secretion of IL-12, and they could not stimulate the T cells in MLR effectively. After stimulation of LPS, they showed a higher expression of MHCII and CD86 and a stronger IL-2 and IFN-γproduction and higher stimulatory capacity of allogeneic T cell.Conclusions GM-CSF at a concentration of 5ng/ml is the best method to generate large number of lewis rat immature dendritic cells, and this make it possible to induce immunological tolerance by DC.Part II: Construction of Adenoviral Vector Encoding Rel-A Gene and Identification of Their ExpressionObjective To choose the Rel-A siRNA with the highest gene silence efficacy from the 4 pairs of siRNA and construct a recombinant Ad containing Rel-A specific siRNA expression cassette.Methods (1)4 pairs of siRNA targeting rat Rel-A mRNA were designed. Combined with control group there were 7 experiment groups altogether. Then those siRNA were transfected into the cultivated rat BRL cells use the way we conformed in part 1. The Rel-A gene expression effectiveness was tested by reverse transcription-PCR and Western blotting. (2) The Rel-A specific siRNA expression cassette was cloned behind the sequence of H1-RNA promoter, and the positive clones containing target gene were selected and appraised. A recombinant adenovirus was produced by a double-recombination event between cotransformed adenoviral backbone plasmid pAdEasy-1 and a linearized shuttle vector. Positive clones were selected and confirmed. Then they were linearized and transferred into the 293A cell to amplification, and then were purified by density gradient ultracentrifuge and titrated. The desired Ad vectors were transfected into the BRL cells to detect the gene silence efficiency.Results (1)The second siRNA was the most efficient one and the Rel-A mRNA expression was decreased 90%, the protein expression was decreased 80% compared with the blank group. (2) Both the plasmid pShuttle-H1-p65 and the recombinant Ad were confirmed correct. The recombinant Ad were generated in a high titer which reached 3.0×1010 pfu/ml, and could efficiently knock down the Rel-A expression in vitro.Conclusions (1)The second pair of siRNA was the most efficient function of gene silence.(2) The recombinant Ad which containing Rel-A specific siRNA expression cassette was successfully constructed.Part III: Effect of Biological Characteristics of Rat Bone Marrow-derived Dendritic Cells Transfected by Ad-shRNA-Rel-A in VitroObjective To study the effect of biological characteristics of rat bone marrow-derived dendritic cells transfected by Ad-shRNA-Rel-A gene in vitro.Methods Recombinant adenovirus expression plasmid Ad-shRNA-Rel-A was transfected into rat bone marrow-derived dendritic cells to arrest their maturation, construct tolerenence DC. The expression of transfected gene was detected by western blotting analysis, surface molecules of Ad-shRNA-Rel-A-DC were detected by FCM. Autologous T cell proliferation stimulated by Ad-shRNA-Rel-A -DC was detected by MTT assay, and the level of IFN-γand IL-10 secreted by DC was analyzed by ELISA.Results Western blotting analysis detected high and stable expression of Ad-shRNA-Rel-A gene in Ad-shRNA-Rel-A transfected DC, and compared with the untransfected DC, DC transfected by recombinant adenovirus vector encoding Rel-A didn't up-regulate the expression of CD86(P>0.05). In contrast to untransfected DC, allogeneic T cells proliferation induced by Ad-shRNA-Rel-A-DC was obviously lower(P<0.01), and a stronger IL-10 secretion level(P<0.01) and a weaker IFN-γsecretion level(P<0.01) was detected in the Rel-A transfected DC.Conclusions Rel-A gene can be highly and stably expressed in Ad-shRNA-Rel-A transfected DC, and it can restrain their maturation. DC transfected by Ad-shRNA-Rel-A and loaded with BN antigen can lower the allogeneic MLR, which provided an experimental base for immunological tolerance induction by DC.PART IV: The establishment of a model of rat orthotopic liver transplantationObjective To establish a reliable animal model of rat orthotopic liver transplantation with modified cuff technique.Methods 120 cases of rat orthotopic liver transplantation were performed by using the two-cuffed technique with some modification in graft harvesting, the anastomoses of suprahepatic vena cava and the perioperative treatment.Results Of the last 30 cases, the mean time of anhepatic period was 12.3min, The 24 hours and 2 week survival rates were 94.3% and 90% respectively.Conclusions The modified two-cuffed technique is convenient and easy to repeat. It can enhance the stability and survival rate of rat orthotopic liver transplantation models. Skilled surgical techniques and shortening the anhepatic phase of recipient as much as possible are the keys to animal survival.PART V: Tolerance in Rat Allograft liver transplantation Induced by Dendritic Cells Transfected by Rel-A GeneObjective To establish rat allograft liver transplantation model of acute rejective reaction and investigate the effect and mechanisms of recipient-derived imDC transfected by Rel-A and loaded with donor antigen to induce immune tolerance. To develop a more applicable approach that uses recipient-derived dendritic cells to induce tolerance for clinical liver transplantation.Methods Liver transplantations were performed from BN or Wistar donors to Lewis recipients. DC were cultured from recipient rats (Lewis) bone marrow with low dose GM-CSF and IL-4. At day 7, they were transfected by Rel-A to arrest maturation, and at day 9, they were plused with BN splenocyte lysate for another 2 days. Then, this modified recipient bone marrow derived DC were injected into recipient rats 7 days before transplantation. Null treatment, DC-treatment, Adv-0 transfected and plused BN spleen cell lysate, and a third party donor (Wistar) were served as controls. The survival of liver allografts were observed, cytokine levels were analyzed by ELISA. Their allostimulatory activity was assessed in vitro by one-way MLR. Pathological examination was performed to identified the grade of rejection.Results Compared with Null treatment, DC- treatment, and the third party donor(Wistar) controls, Rel-A transfected DC plused with BN splenocyte lysate markedly prolonged the survival of liver allografts in an antigen-specific manner(26.8±1.76d, P<0.01). Rel-A-DC loaded with BN antigen elicited markedly lower proliferative responses and reduced IL-2 and IFN-γproduction. In contrast to other groups, Rel-A-DC loaded with BN antigen show a lower grade as estimated by pathological examination.Conclusions Successfully and surely established rat allograft liver transplantation model of acute rejective reaction. Recipient-derived imDC transfected by Rel-A and loaded with donor splenocyte lysate could induce immune tolerance in a donor-specific manner, it might be associated with induction of T-cell hyporesponsiveness and enhanced T-cell apoptosis. Our successful induction of tolerance by DC in a recipient manner provides a more feasible strategy for deceased-donor liver transplantation.
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