The Effect Of Mesenchymal Stem Cells On Prolong Cardiac Allograft Survive With Immune Tolerance

At present, organ transplantation becomes the preferred therapy for a lat ofend-stage disease. For the recent 50 years, it has achieved great success and broughtimproved life qualities to numerous patients. With the development ofimmunogenetics, we have increasing knowledge on immunological mechanismsunderlying transplantation rejection. The application of CsA to clinic greatly reducedthe morbidity of rejections. Nevertheless, clinical transplantation still faces someproblems such as lifelong immunosupression associated with toxicity, opportunisticinfection and a high incidence of cancer. Donor specific organ transplantation immunetolerance, being definede as no medication(only routin immunosuppressive agent), norejectiong in MHC mismatched organ transplantation, is the trend of organtransplantation.Mesenchymal stem cells (MSCs) may be derived from adult bone marrow, fat,and several fetal tissues. In vitro, MSCs can be expanded and have the capacity todifferentiate into several mesenchymal tissues, such as bone, cartilage, and fat. Theyescape the immune system in vitro, and this may make them candidates for cellulartherapy in an allogeneic setting. They also have immunomodulatory effects, inhibitT-cell proliferation in mixed lymphocyte cultures, prolong skin allograft survival, andmay decrease graft-versus-host disease (GVHD) when cotransplanted withhematopoietic stem cells. Recent studies showed that MSCs possessedmmunomodulatory potential. So it may be used to induce immuno-tolerance in organtransplantation. In the study, we isolated MSCs from rat bone marrow and establisheda model of heterotopic rat heart transplantation to research the effect of toleranceinduction with MSCs on rat heart transplantation and to explore the underlyingmechanism. PartⅠIsolation Purfication Rification And Identifcation Of Rat Bonemarrow-Derived Mesenehymal Stem Cell.【Objective】: To explore a method for the isolation and purification in vitro ofmesenchymal stem cells (MSCs) from rat bone marrow and analyse theirphenotypical properties. To investigate the possibility of using mesenchymal stem cellon allograft transplantation【Methods】: MSCs were isolated and purified from the bone marrow of SD rat s bydensity gradient centrifugation and marked with DAPI before transplatation. 12 maleWistar rats (body weight: 200g-250g) were separated randomly into 2 groups.Group A was control group (n=6). Group B was MSCs transplatation group (n=6): wetransplant the exogenous MSCs into Wistar. Every rats received 1×10~6 MSCs/1ml,and these MSCs were injected from vena caudalis. 1 weeks later, we used the methodsof fluorescence microscope to investigate the survival MSCs.【Results】: The isolated MSCs were mononuclear cells in marrow. Their livingbehavior was quite stable in L-DMEM containing 100ml/L newborn bovine serum,and exhibiting a large expansive potential and the typical fibroblast-like morphology,and uniformly expressing CD90, unexpressing CD34, CD45. 1 weeks later, wefound survival MSCs on bone marrow slides in MSCs transplatation group.【Conclusion】: A method for the isolation and purification in vitro of MSCs fromrat bone marrow has been established, which exhibit s expression of CD90,unexpressing CD34, CD45. Purified MSCs had its own distinctive immunogenicity,exogenous MSCs transplantation did not need immune inhibitor previously,exogenous MSCs can survive, home, and differentiate into vascular endothelial cellsin host without immunologic rejection reaction.PartⅡMixed Lymphocyte Culture (MLC) with MSCs【Objective】: To investigate the immunogenity and immune regular ability of mesenchymal stem cells (MSCs) derived from bone marrow through mixedlymphocyte culture.【Methods】: MSCs and mixed lymphocyte reaction (MLR) cultures were set up.Splenic lymphocytes from SD rat were used as activate cells and splenic lymphocytesfrom Wistar rat as reactive cells. The study was composed as 6 groups. GroupⅠ, thecontrol group, was mixed culture of 1×10~5 activate cells and 1×10~5 reactive cells.GroupⅡwas mixed culture of 1×10~4 MSCs from SD rats and the same amount ofreactive cells as GroupⅠ. GroupⅢwas mixed culture of 1×10~4 MSCs from SD ratsand the same amount of active and reactive cells as GroupⅠ. GroupⅣis mixedculture of 1×10~4 MSCs from SD rat and the same amount of active and reactivecells as GroupⅠplus 1-methyltryptophan (1-MT). GroupⅤis mixed culture of1×10~4 EPC from SD rat and the same amount of active and reactive cells as GroupⅠplus phytohemagglutinin (PHA) as a stimulator. GroupⅥis mixed culture of 1×10~3MSCs and the same amount of active and reactive cells as GroupⅠ. Each group wascultured for 120h. The number of lymphocyte in each group was calculated withliquid scintillation counter.【Results】: MSCs inhibited T-lymphocytes proliferation of MLR cultures in adose-dependent manner. The content of tryptophan decreased greatly in MSCs andmixed lymphocyte reaction (MLR) cultures. 1-MT reversed the effect of MSCs..【Conclusion】: MSCs could suppress T21ymphocyte responses via IDO enzymeactivityPartⅢEstablishment of Heterotopic Heart Transplantation Model in Rats【Objective】: To establish the methods of allograft heart transplantation in rats.【Methods】: After anesthesia and heparinization, SD rat heart was harvested as donor.The aorta of donor heart was anastomosed to the abdominal aorta of Wistar rat asrecipient with end to side fashion, and donor pulmonary artery was anastomosed to inferior vena cava of the recipient with the same fashion. The animals were dailyinspected and the survival days were counted.【Results】: After short time training, the transplantations were totally successful.【Conclusion】: The heterotopic heart transplantation model we established wasconvincable as a model of allograft heart transplantation.PartⅣEffect of Acute Rejection of Allograft Heart Transplantation in Rats by Donor MSCs【Objective】: To explore the effect of donor MSCs on inhibition of acute rejection inheterotopic allograft heart transplantation in rat..【Methods】: 64 Wistar rats as recepients were divided into four groups randomizely.The animal transplantation model in Part 3 was used. In GroupⅠ, there was no MSCsinjection and CsA feeding. In GroupⅡ, recepients received 1×10~6/1ml MSCs vialumbar vein in implantation. In GroupⅢ, rats were fed with the CsA after transplantfor 7days. In GroupⅣ, recepients received 1×10~6/1ml MSCs via lumbar vein inimplantation and fed with the CsA after transplant for 7days. Donor heart survivaltime was viewed by abdominal touch. On the seventh day after transplantation, therats were sacrificed and the spleen were taken out for CD4 and CD8 T cell countingand CD4/CD8 ratio; allograft hearts were extracted for pathological study; Level ofserum IFN-γand IL-10 was detected by ELISA assay.【Results】: The surviving time of transplanted hearts in groupⅠto groupⅣwas7.5±0.5 days, 14.8±2.1 days, 23.4±1.3 days and 36.6±4.2 days respectively.Statistical analysis showed that it was longer in groupⅡthan that in groupⅠ(p＜0.05)and it was longer in groupⅣthan that in other groups (p＜0.01). There was lesserpathological changes in groupⅣthan that in other groups. CD4/CD8 ratio inperipheral blood of recipients was higher in groupⅡ, groupⅢandⅣthan that ingroupⅠ(p＜0.05); It was higher in groupⅣthan that in other groups(p＜0.05). SerumIFN-γlevel of recepients was lower in groupⅡand groupⅢthan that in groupⅠ(p＜0.05); It was lower in groupⅣthan that in other groups(p＜0.05). Serum IL-10 level of recepients was higher in groupⅡ, groupⅢand groupⅣthan that in groupⅠ(p＜0.05); It was higher in groupⅣthan that in groupⅠand groupⅡ(p＜0.05). Therewas no significant difference between groupⅣand groupⅢ(p＞0.2).【Conclusion】: Intravenous administration of bone marrow derived MSCs alleviateimmunorejection in heterotopic rat heart transplantation. The mechanisms involve thesuppressive effect of MSCs on lymphocytic proliferation and modulation on cytokineserection.