Research Of Inducing Transplantation Immune Tolerance By Porcine Bone Marrow Mesenchymal Stem Cells

[Background]Orthotopic heart and lung transplantation has become a effective method about the treatment of end-stage heart and lung diseases,which has opened up a new field for medical science.At present,the donor heart and lung preservation, surgical technique and postoperative infection is no longer handicap of the successful heart transplantation.However,immune rejection after transplantation is a serious threat to the long-term survival of transplant patients.Donor shortage has plagued surgeons.Induce donor-specific immune tolerance can effectively solve this problem. Moreover,most experiments are carried out on rodents,but crucial prerequisites for the development of safe pre-clinical protocols in biomedical research are needed through suitable large animal models.In transplantation particularly,experimental minipig models have developed dramatically.Mesenchymal stem cells are adult stem cells in bone marrow,with support hematopoiesis,tissue repair,differentiation potential and the potential immunological characteristics.Diannan minipig has become the most potential of experimental animal researched in human disease model, especially in heart and lung transplantation research.Recent studies show that the human and rodent MSCs in vitro can inhibit the proliferation of T lymphocytes, treatment of graft versus host disease,prolong skin graft survival time.However, mechanism of immunoregulation and inducing transplantation tolerance in vivo are uncertain.Migrating,transformating,homing to the target organ and security of application of MSCs in vivo are dubious.Not seen the study report about immunological characteristics of pBMSCs.Therefore,this study is to research pBMSCs biological characteristics through the establishment system of isolation, culture expansion of pBMSCs.Establishing mixed culture system in vitro and infusing pBMSCs in vivo is to research the mechanism of the immunological characteristics of pBMSCs.[Methods](1)To isolate pBMSCs through the whole bone marrow adherent means. Morphological identification of pBMSCs can be carried out through inverted microscope and transmission electron microscopy.Molecular phenotype pBMSCs can be carried out through Flow Cytometry.Potential differentiation Identification of pBMSCs can be carried out through inducing pBMSCs to differentiate osteocyte and adipocyte.(2)The system of mixed lymphocyte culture will be established.The inhibitory effect of pBMSCs on peripheral blood lymphocytes(PBLs) can be detected through mixed lymphocytes culture test(MLC).The changes of Foxp3 gene and cytokines interferon-gamma(IFN-γ),transforming growth factor beta(TGF-β) can be detected through Real-Time quantitative polymerase chain reaction(Real Time-PCR) after mixed lymphocyte culture.(3)The changes of peripheral blood T lymphocyte subsets can be detected through Flow Cytometry post-infusion pBMSCs.[Results](1)Through the whole bone marrow adherent means we obtained high purity and high activite primary pBMSCs.Received pBMSCs were cultured 20 passages in vitro,our data demonstrated the evidence that their biological characteristics were stability:Morphological identification show pBMSCs are fibroblast-like cells;flow cytometry identify show pBMSCs are stable express CD29, CD44,CD105,did not express CD45,SLA-DR;identification of pBMSCs pluripotent ability show it can differentiate into osteocytes and adipocytes.In this study,high purity and high activity primary pBMSCs were obtained through the whole bone marrow adherent means,and a pBMSCs lines were obtained through transfer of culture.The system of isolation,culture expansion of pBMSCs was established.All these provided the seed cells for the next researching of pBMSCs' immunological characteristics and pBMSCs' inducing immune tolerance.(2)The system of mixed lymphocyte culture is established in this study.The pBMSCs inhibit significantly proliferation of peripheral blood lymphocytes and proliferation of peripheral blood lymphocytes stimulated by PHA.However,pBMSCs supernatant not inhibit proliferation of peripheral blood lymphocytes,Even stimulate the proliferation of peripheral blood lymphocytes.The high expression of Foxp3,TGF-βand IFN-γis on PBLs mixed culture.IFN-γexpression was significantly lower than Foxp3 and TGF-β. (3)The expression of Foxp3,TGF-βand IFN-γof peripheral blood lymphocytes were significantly increased in the post-infusion pBMSCs 24h,increased along with the time and went to the peak after 72h,began to decrease after 96h(P＜0.05).Peripheral blood T lymphocyte subsets were no significant difference.[Conclusion](1)In this study,high purity and high activity primary pBMSCs were obtained through the whole bone marrow adherent means,and a pBMSCs lines were obtained through transfer of culture.The system of isolation and culture expansion of pBMSCs was established.All these provided the seed cells for the next researching about immunological characteristics of pBMSCs and inducing immune tolerance by pBMSCs.(2)The proliferation of peripheral blood lymphocytes were inhibited significantly by pBMSCs in vitro mixed lymphocyte culture.The pBMSCs not stimulate alloimmune response,but can regulate the immune system in vivo.(3)The mechanism of pBMSCs with low immunogenicity and immunomodulatory properties is associated with interaction between soluble cytokines,inducing high expression of Foxp3 in regulatory T cells and the direct contact between cells.The foundation was layed for the next research of inducing immune tolerance in Diannan minipig heart and lung transplantation by pBMSCs.(4)A robust and permanent immune tolerance will be induced expectably by pBMSCs in Diannan minipig heart and lung transplantation before long.