| ObjectiveEstablished the methods of culturing,derivating and amplificating denfritic cells in vitro from bone marrow stem cells of DA rats and established the modified rat model of orthotopic liver transplantation and the rat liver transplantation model of acute rejection,to investigate the survival time of allograft,this research modifying imDC with hIL-10 gene.Methods1. DC were cultured ,derivated and amplificated in vitro from bone marrow stem cells of DA rats.After their morphology,immunophenotype and function were identified and confirmed,the plasmids of pIRES2-EGFP-hIL-10 were transfected into imDC to detect expression of hIL-10 gene.2. Established the modified rat model of orthotopic liver transplantation and the rat liver transplantation model of acute rejection: On the basis of the previous two-cuff-technique,120 cases of rat orthotopic liver transplantation were performed with fast resecting the donor liver,continuous sutureing suprahepatic inferior vena cavum and stoping bleeding point with hot pin and so on and to observe the survival rate and viability.3. One hundred and forty cases of rat allogeneic orthotopic liver transplantation were accomplished in the strain combination of DA to Lewis rats,which were divided equally into 5 groups,including non- injection group(control group),mDC group, imDC group,empty vector group and hIL-10 group.Then 2×106 cells were respectively intraperitoneally injected to Lewis rats with distinct cells,such as mDC, imDC,empty vector-transfected imDC and hIL-10-modified imDC.Four Lewis rats from each group were randomly sacrificed on days 3 , 7 and 10 after transplantation.Hepatic function (ALT and BLIL) and cytokines alteration were evaluated in peripheral blood.The pathological changes of the liver graft were observed.And the DNA fragmentaion characteristic of apoptosis in spleen. Survival analysis were performed for remnant rats, except rats dead for technical factor.Results1. The process had been successfully established for culturing,derivating and amplificating DC from bone marrow stem cells of rats.These DC have been validated and identified by the inverted microscope,transmission electron microscope, scanning electron microscope and flow cytometer.The outcome of morphology showed that DC has irregular shape,the unsymmetric nucleus,and slender dendritic processes on cell surface.The result of immunophenotype demonstrated that on the cell surface of mDC and imDC,the expression level of OX62 were both highly;however, the expression level of costimulatory molecules,CD86, on imDC surface was lower than that on mDC surface.The consequence of mixed lymphocyte reaction( MLR ) displayed that the capability of imDC stimulating allogeneic T lymphocytes was significantly weaker than the ability of mDC's( P=0.0005 ).The sequences of hIL-10 in pIRES2-EGFP- hIL-10 was consistent with those in Pubmed Genebank, which has been confirmed by gene sequencing;and the PCR notified the clear zone in 540bp.After transfection,the imDC could emit green fluorescence under fluorescence microscope,the CD86 and CD80 expression on the imDC surface did not be obviously impacted in the result of flow cytometry.The result of MLR demonstrated that the imDC induced lower T-cell proliferatoin in hIL-10 group(P=0.024,P=0.033).The result of ELISA showed that the expression of hIL-10 in hIL-10 group was markedly higher than that in other groups(P=0.004,0.004 and0.003).And the Westen-Blot had detected the expression of IL-10.2. We have been succeed in establishing the modified rat model of orthotopic liver transplantation and the rat liver transplantation model of acute rejection .3. There was only a slight number of mononuclear cell infiltration in hepatic graft after orthotopic liver transplantation in IL-10 group , which belonged to a"non-identified"or"slight"type of AR with RAI score 2 to 5;and the liver graft showed up regeneration of hepatocyte along with a small number of neutrophile granulocyte infiltration at day 10.At day 7 and 10,liver function of IL-10 group began to recover,but compared to those of imDC group and empty vector group , the difference was not statistically significant. The outcome of ELISA notified that at day 7, the serum levels of IL-12 in IL-10 group is lower compared to those in control group, mDC group,imDC group and empty vector group(P=0.000,0.000,0.005 and 0.008).The MST of rat recepients in IL-10 group was 40 days that was longer than imDC group ( 23 d ) ( P = 0.002) and empty vector group( 25 d ) ( P = 0.002 ) .Conclusions1. A large number of DC could be developed,induced and amplificated in this experiment by combination of separating medium of rat lymphocytes and maneuver screening.After transfection,hIL-10 gene could be effective expression, and didn't impact imDC's phenotype and could induce significantly low allogeneic T lymphocyte reactivity of imDC in MLR.2. The modified methods shorten the operation time in rat orthotopic liver transplantation model,and enhanced the stability and increased survival rate of rat orthotopic liver transplantation model.It can meet the requirements of experiments.3. hIL-10 gene-modified imDC could induced long-term allograft survival of receptors in allogeneic rat liver transplantation than the control group. |
