Ibhibition The Expression Of CD80/CD86 Gene Of Dendritic Cells Induces Immune Hyporesponesiveness In Rat Liver Transplantation

The treatment of end-stage liver disease is difficultand the effect is poor.Liver transplantation has been accepted for the advanced liver disease patients.The rejection after liver transplantation is less severe than in cardia,lung,pancreas and kidney,but there still have a series of rejection problems which are eager to be solved[1].At present,although many new immunosuppressive drugs are used to decrease the incidence of rejection,the immunosuppressive agents for long-term and lifelong applications not only aggravateeconomic burden,but also cause the immune function decreassing,and may induce infection,promote the recurrence of hepatitis and tumor,which has brought a very negative impact on transplant patients.Therefore,people are trying to find another effective methods,so that the transplanted organs can survive without the use of immunosuppressive agents.The most ideal method to solve this problem is to induce the specific immune tolerance to the allograft.The immune recognition,activation and proliferation of T cells to the donor antigens are the basis of cell immunity of organ transplantation.The recognition of T cells to antigens need the participation of antigen presenting cell.Dendritic cell(DC)is the most important APC in organism,which is the only one that can activate initiated T cells(T cells APC),and studies have confirmed its effect of activation on Tr[1,2].In fact,compared with Tr,DC was found to induce tolerance in liver transplantation,which is considered to be a role of inducing tolerance[3].T cells play a key role in allograft rejection.The recognition of T cells to the antigens of donor includes direct and indirect recognition.At present,more and more scholars believe that the indirect recognition pathway of T cells plays an important role in the pathogenesis of chronic graft dysfunction.Therefore,it is more worthy to focused on the molecular biology mechanism of blocking indirect recognition pathway in inducing immune hyporesponesiveness and immune tolerance of transplantation.The activation of T cells in the indirect recognition pathway requires the combination of the co-stimulatory molecules on the surface of the antigen presenting cells and the corresponding receptors on the surface of T cells,and the CD80/CD86-CD28 is the most important one.To this end,we used the advanced RNA interference technology to inhibit the expression of CD80 and CD86 on the surface of dendritic cells of donor and Collected the experimental results in vitro and in vivoto observe the effect of blocking the indirect recognition pathway of T cells to allogene liver transplantationof rats,and studed its related mechanism preliminary.Part ICulture and Identification of Rat Myeloid Dendritic Cells in VitroObjective:To aquire rat myeloid dendritic cells in three different mature states(imDC,smDC and mDC).Methods:rrGM-CSF and rrIL-4 were used to induce bone marrow derived mononuclear cells differentiating into dendritic cells(imDC)in vitro.Stimulated with tumor necrosis factor-a(TNF-a)to obtain semimature dendritic cells(smDC),simultaneously stimulated with Lipopolysaccharides(LPS)to obtain mature dendritic cells(mDC).Three kinds of dendritic cells were identified by morphology,expression of cell surface markers and cytokines,the activation of allogeneic lymphocytes and CD4+CD25+Treg.Results:smDC showed a kind of intermediate developmental state of DC cells in form.It expressed MHC-? in a high level,meanwhile,it expressed CD80/86 in a certain degree,which was more close to imDC.However,there was no activation of allogeneic lymphocytes in imDC and smDC,but the ability of smDC to activate CD4+CD25+Treg proliferation was stronger than imDC.Conclusion:imDC,smDC and mDC in high purity and in a stable functional state can be obtained by using rrGM-CSF and rrIL-4.Part IIRNA Interference Blocks the Expression of CD80/CD86 Gene in Different Mature States of Dendritic Cells of RatsObjectives:To construct the lentiviral vector of RNAi which aiming at CD80 and CD86 gene of rat,and to observe it's effects on rat myeloid dendritic cells in vitro.Methods:According to the target sequence of RNAi which aimed at CD80 and CD86 gene of rat,we constructed the lentiviral vector.Different mature states of dendritic cells(imDC andsmDC)in vitro were infected by it.The infection efficiency were detected by fluorescence microscope.The expression level of CD80 and CD86were detected by flow cytometry,and the expression of CD80 and CD86 related proteins were detected by Western Blot.Results:The result of PCR of recombinant plasmid and sequence confirmed that LV-GFP-shCD80 and LV-GFP-shCD86 were constructed correctly.The most suitable MOI value of infection were 20,and the infection rates were 79.98%±5.01%and 81.38%±4.96%in imDC and smDC respectively.The result of flow cytometry pointed out that,after plusing LPS to promote maturation,the expression of CD80 and CD 86 on imDC and smDC which infected by LV-GFP-shCD80 and LV-GFP-shCD86 both were lower than which in the blank control group,and there were statistically significant differences(p<0.01).At the same time,the expression of OX62 and MHC-II were not affected.It was recommended in Western Blot that the expression of CD80 and CD86's protein on imDC and smDC which infected by lentiviral vector of RNAi were significantly lower than that of the negative control group,and the protein expression of imDC group was lower than that of smDC group.Conclusions:The lentiviral vector of RNAi which aimed at CD80 and CD86 gene of rat is constructed successfully,and it can inhibit the increase of expression level of CD80 and CD86 in imDC and smDC after plusing LPS to promote maturation.Part ?Donor DCs Pretreated by Recombinant Lentivirus Blocking Costimulatory Pathway in VitroObjectives:To observe the immune reactivity of DCs pretreated by recombinant lentivirus In vitro,and to approve they can induce immune tolerance in transplantation,to provide theoretical basis for next experiment in vivo.Methods:We isolated the T cells in the recipient splenic,and progressed mixed lymphocyte experiment,then we observed the ability of donor's dendritic cells pretreated by recombinant lentivirus to stimulate the proliferation of recipient T cells.Sensitize receptor in advance with each DCs,and progressed mixed lymphocyte reaction again,which included the sensitized T cells in the recipient splenic and the DCs pretreated by recombinant lentivirus.the immune activity of the sensitized T cells of recipients for donor specific antigen were detected.At the same time,we tested the ability of DCs-induced proliferation of recipient Treg in the twice mixed lymphocyte reaction through flow cytometry.Results:The imDC and smDC,pretreated by recombinant lentiviral vector of RNAi which aimed at CD80 and CD86 gene of rat restrained the proliferation of recipient T cells obviously.And they induced specific low immune response against donor antigen.Those abilities of imDC were stronger than smDC.The DCs pretreated by recombinant lentivirus induced proliferation of recipient Treg,and the ability of smDC were stronger than imDC.Conclusion:The DCs pretreated by recombinant lentivirus can induce immune tolerance in transplantation in vitro.Part IVThe Building of Acute Rejection Model after Rats Orthotopic Liver TransplantationObjectives:To establish a stable models of rats orthotopic liver transplantation and observe the characteristics of acute rejection on Lewis to BN.Methods:On the basis of a large number of training of SD rat liver transplantation,we used modified "two sleeve tube method" to establish a stable models of rats orthotopic liver transplantation.In the formal experiment,we used Lewis rats as donors and BN rats as receptors,which were divided into the group of BN to BN,the non-intervention group of Lewis to BN and the intervention group of Lewis to BN(feeding treatment dose of rapamycin in the perioperation).We Observed the postoperative survival conditions and survival time of rats,and detected the changes of liver function postoperative 1 d,4 d,7 d,10 d,then carryed on general observation to liver and surveed the pathological changes.Results:The rats in group of BN to BN survived for a long time.There was no obvious deterioration on liver function,and no obvious rejection or only had slight rejection in pathological observation in the whole period of time.The rats in non-intervention group of Lewis to BN were dead in early postoperative.Their liver function deteriorated sharply in early postoperative,and there was rapid and strong rejection in pathological observation.The rats in intervention group of Lewis to BN were dead after drug withdrawal,and all rats were dead in two weeks of postoperation.Their liver functionwas recovered gradually,but deteriorated rapidly after drug withdrawal,and there was varying degrees of rejection in pathological section.The rejectionwas slightly before drug withdrawal,but deteriorated rapidly after drug withdrawal.Conclusion:Stable rejection appears obvious after liver transplantation in the group of Lewis?BN,it can be used as acute rejection model in ROLT.Rapamycin can restrain the rejection in the group of Lewis?BN effectivly.Part VExpression of CD80/CD86 Gene Blocked by RNA Interference in Different Mature States of Dendritic Cells Induces Immune Hyporesponesiveness in Rat Liver TransplantationObjective:On the basis of the allogenetic rat liver transplantation model,to discuss its feasibility and immunological mechanisms of donor dendritic cells processed by recombinant lentivirus induced immune hyporesponesiveness in vivo.Methods:We used Lewis rats as donors and BN rats as receptors,the rats were divided into immature dendritic cells group,semimature dendritic cells group,interferenced immature dendritic cells group and interferenced semimature dendritic cells group.During the seven days before transplantation,we infusied different dendritic cells to pretreat rats via tail vein.Then we carryed out two sleeve tube orthotopic liver transplantation in rats.To observe survival conditions and survival timewe detected liver function,cytokine levels and Treg cells proportion in peripheral blood and histopathological changes in the liver in 1 d,4 d,7 d,10 d respectly after operation.Results:Compared with the simple use of rapamycin,infusing different dendritic cells from donor preoperation prolonged the survival time of receptors after operation,and reduced the deterioration of liver function.The effections were particularly evident after drug withdrawal 10 days).On the basis of the use of rapamycin,infusing different dendritic cells from donor preoperation inhibitedthe secretion levels of cytokines of Thl type(IL-2 and IFN-y)and promoted the secretion levels of cytokines of Th2 type(IL-4 and IL-10),and induced the producing of Treg cells in the peripheral blood.In all kinds of reactions above,RNA interference CD80/CD86 groups were better than the non-interference groups.Conclusions:On the basis of the use of rapamycin,infusing different dendritic cells from donor preoperation can induce immune hyporesponesiveness.Two measures may have a synergistic effect.The costimulatory pathway blocked by RNA interference can protect the graft in vivo.Through the experiment in vivo and in vitro,we confirm that the imDC and smDC of donor can induce immune hyporesponesiveness of receptor.RNA interference can block the expression of costimulatory molecules CD80/CD86 on different mature states of DCs.The ability of inducing immune hyporesponesiveness of DCs recepted RNAi is enhanced.It points out a research direction in inducing immune tolerance.