| BackgroundBurns, war trauma can lead to severe tissue or limb defects, due to the lack ofautologous tissue, patients often suffer different levels of somatization disorder andpsychological burden. Composite tissue allotransplantation (CTA) can provide satisfactoryappearance and functional reconstruction, which has been increasingly appreciated both athome and abroad. However, the risk of opportunistic infections and malignancies causedby long-term post-transplant immunosuppressant, as well as the potental inevitablechronic rejection impede its wide clinical application. Therefore, there are urgentrequirement for exploring a new anti-graft rejection protocol and induce a donor-specificimmune tolerance.Bone marrow transplantation has been proved to be effective for tolerance induction,which has been confirmed in the laboratory during the liver, kidney, heart and other solidorgan transplantation, and is expected to solve clinical organ transplant rejections. It hasbeen suggested that vascularized bone marrow transplantation (VBMT) may be superior toconventional bone marrow cell transplantation for tolerance induction. This phenomenonis thought to be associated with the bone components, which serves as a vascularizedmethod of delivering donor-origin stem and progenitor cells. However, the exactmechanism remains poorly understood. This remind us that whether the allogeneic hind limb graft, a form of CTAs involving the bone components, can induce tolerance throughVBMT, and what is the underlying mechanism?Bone marrow mesenchymal stem cells (BMSCs) are a group of self-renewalnon-hematopoietic stem cells with wide biological functions. BMSCs express low levelsof MHC I molecules and MHC II molecules as well as coactivator molecules such asCD40/CD80/CD86. These properties make BMSCs low immunogenicity and can betransfused between different strains, species without the consideration of rejection by therecipient immune system. One of the prominent properties of BMSCs is theimmunomodulatory property, which has been shown effective in a series of immunedisorder diseases and hold potential promise in the treatment of transplant rejection.Based on the fact that there are no studies about the possibility of limb allograft servingas vascularized bone marrow transplant inducing immune tolerance as well as few reportsabout BMSCs applicated for limb allotransplantation, in this study, we intend to study thepossibility of hind limb serving as VBMT and its effect for chimerism induction and theallograft survival, and further to explore the role of BMSCs for transplanted limb survival.The mechanism will be investigated as extensive as possible.Materials and Methods1st. We intend to establish hind limb transplant model and the bone-removal hind limbtransplant model. With the intact hind limb model as control, we will investigate whetherthe bone-removal process will affect the blood supply of the bone-removal hind limb andthe subsequent graft survival. At the presence of CsA, the graft survival will be monitoredin all experimental group, and the role of bone components for graft survival will beobserved.2nd. Forty-eight hind limb transplants were performed. Isograft transplantations wereperformed in groups1and2between genetically identical LEW rats (IsotransplantationGroups). Allograft transplantations were performed between BN rats and LEW rats ingroups3to6. One milli liter of N.S was given to rats in groups3and4(RejectionGroups). In groups5and6(Experimental Groups), all recipients were treated with tapered CsA, which was administered on day0at a standard dose of16mg/kg per day for1week,tapered to2mg/kg per day over4weeks, and maintained at this level thereafter. Thefemur, tibia, and fibula were kept intact in groups1,3, and5(intact grafts), but they wereremoved in groups2,4, and6(bone-removal grafts, B-R grafts) before transplantation.The survival as well as the chimerism level was dynamically recorded, the role of Treg fortolerance induction was also investigated.3rd. Different concentrations of IFN-γ were added to BMSCs medium to evaluate itseffect on immune regulatory property of BMSCs. IFN-γ pretreated BMSCs were furtheradministrated intravenously to the hind limb recipient, followed with observation of thegraft survival.4th. The exact mechanism of IFN-γ pretreatment for BMSCs proliferation and its immuneregulatory property were investigated. With the application of mixed lymphocyte reaction,the role of IFN-γ pretreated BMSCs for T cell activation, proliferation, apoptosis and Treggeneration was also investigated.Results1st. The limb transplantation models including intact hind limb model andbone-removal hind limb model were successfully established in rats. The bone-removalhind limb model serving as counterpart of intact hind limb model was confirmed feasiblefor investigation of role of bone components for the hind limb survival. We demonstratedthat with tapering CsA treatment, the intact hind limb long-term survived, while thebone-removal hind limb was rejected at the same CsA regime, revealing the important roleof bone components for hind limb survival.2nd.Peripheral chimerism was observed as a phenomenon in all allogeneic hind limbreceipient, myeloid chimerism was only observed in receipient rats receiving allogeneichind limb followed with CsA treatment. With the rejection reaction, peripheral chimerismobserved in other groups diminished. Subsequent to the myeloid chimerism, donorspecific regulatory T cells (Treg) were observed in receipient rats receiving allogeneichind limb followed with CsA treatment, which was demonstrated to be responsible for the hyporeactivity of receipient for donor antigens. These results revealed that immunetolerance may be achieved in allogeneic hind limb through chimerism induction byVBMT.3rd. IFN-γ pretreatment triggered the immune regulatory property of BMSCs in a dosedependent manner. IFN-γ treated BMSCs prolonged the survival of hind limb allograftwhen administrated to the receipients.4th. IFN-γ treated BMSCs showed an elevated expression of membrane moleculePD-L1, through which the T cell proliferation was inhibited with an increased proportionof T cell apoptosis and Treg generation in the MLR.ConclusionsOur results demonstrated that allogeneic hind limb is superior than other compositetissue allografts (CTAs) for immune tolerance by chimerism induction through VBMT.Meanwhile, IFN-γ treated BMSCs hold promise for immune tolerance establishment byinhibition of T cell proliferation and generation of Tregs through elevated membranemolecule PD-L1. |
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