Experimental Research On Immunological Tolerance Induced By STNFRI-IgGFc Gene Modified Dendritic Cells In Rat Lung Transplantation

Objection1. To approach rat orthotopic left lung allograft transplantation model based on the“three cuff technique”, which will provide a basic model for further research ofimmunological tolerance in post-lung transplantation.2. To explore the effective method of isolation,culture,and proliferation of Wistar ratbone-marrow derived dendritic cells(DCs) in vivo.And to observe the effect ofsTNFRI-IgGFc gene on the differentiation and maturation of DCs.3. To explore the effect of sTNFRI-IgGFc gene modified DCs(imDCs) transfusionon rat orthotopic allograft survival and its possible mechanism.Methods1. We established rat orthotopic left lung allograft transplantation in which theanastomosis of pulmonary artery, vein and bronchus were performed by using thecuff technique under surgery microscope and evaluated the possibility of themodel by achievement ratio of operation, time of operation, and pathologicchange.2. Wistar rat bone-marrow derived dendritic cells were cultured with recombinant ratgranulocyte-macrophage colony-stimulating factor(rr-GM-CSF) and recomb-inant rat interleukin-4(rr-IL-4). At the fifth day,gather DCs and transfectionplasmid-sTNFRIIgGFc in it.And stimulated with lipopolysaccharide (LPS) onDay7.DCs were identified on Day9.morphology of DCs was observed bymicroscope.Surface phenotypes of DCs were flow-cytometrically detected to determine theexperession of maturation markers,including MHC class II,CD80and CD86.DCswere co-cultured with T cells form receipient rat and mixedlymphocyte reaction(MLR) was assessed by MTT method.Supernatants wereanalyzed for the level of production of TNF-a and IL-12by using enzyme linkedimmunosorbent assay(ELISA).3. Lung allograft from SD rat transplanted to Wistar rat.Thirty health adult male ratreceipient rats were randomized into3groups.control group,imDCs group andsTNFRIIgGFcDCs group.The recipient rats in first group were given injection ofPBS100ul,the second were given injection of imDCs in the amount of1x106(dissolved in100ul PBS) and last were given injection of sTNFRIIgGFcDCsin the amount of1x106(dissolved in100ul PBS).all above groups should executethe injection at the operation day,7day before and after operation.Five recipientrats in the each group were killed to make related tests,including MLR,bloodserum were analyze-d for the level of IL-12,IFN-γ,IL-4,IL-10by ELISA.Beside,we continued to observe the survival time of remaining recipient rats.Result1. There are25rats which were operating the left lung transplant.20of them survivein the experiment, and5rates died. so the success rate is80%. The time from lungirrigation to obtain is10±1min, the time of casing in vitro is10±1min, the timeof anastomosis artery, vein and bronchus is40±2min. Do the clipping experimenton transplant recipient (n=5), blocking the right hilar, keep one lung ventilation20min, the heart and the breath of the rats are smoothly. These rats still keep alive in7days.2. The obtained DC cells under light microscope and electromicroscope all showedas a typical DC morphological,and flow-cytometric instrumentation detectionshow th characteristics of DC too. sTNFRIIgGFc-DC in the cell phenotype andMLR have not mature feature of DC in the cytokine secretion, they can secretelarge amounts of sTNFRI, also can antagonize the LPS recipiening effect.3. The proliferation ability of sTNFRIIgGFc-DC group stimulating T cell is lowerthan that in the control group and imDC group (P <0.05), while IL-4, IL-10 4. levels were significantly higher than that in the control group and imDC group (P<0.05), IL-2, IFN gamma levels are lower than the other two groups (P <0.05);TNF alpha in gene transfection group is lower than the other two groups (P <0.05). In histological sections, we can also see sTNFRIIgGFc-DC group ofinflammatory response to light.Conclusion1、 According to the "three cuff "method, it is establish a stable model of ratorthotropic left pulmonary allograft transplantation.2、 Auccessful establish in vitro amplification of rat bone marrow-derived dendritecells, and developed have high fight LPS mature sTNFRI-IgGFc gene which can"the tolerance" DC cells by liposome transfection the plasmid sTNFRI-IgGFcgene.3、 sTNFRI-IgGFc which gene-modified dendrite cells can induce rat lung allografttolerance and prolong allograft survival. Possible mechanism forsTNFRI-IgGFcDCcells could be expression of CD80and CD86. This kind of cellmay leading to T-cell activation signal loss and resulting in immune deviationrelated. It is may alsorelated to sTNFRI-IgGFc, efficient blocking, closedreceptor in vivo activity of TNF-α.