Intra–bone Marrow - Bone Marrow Transplantation(ibm-bmt) Combined With Mesenchymal Stem Cells (mscs) Co-transplantation Induce Immune Tolerance To Skin Allografts

【Background】At present, the main difficulty to cure the large area and severe burn-injured patients is lack of autologus skin, so we can not close the wound with autologous skin graft at early stage. And the skin allograft transplantation can only cover the wound temporarily but not for long term survival, result in infection, sepsis, even multiple organ dysfunction syndrome (MODS) and death. These problems have severely restricted the development and improvement of curing the large area and severe burn-injured patients. Thus, how to induce forever immune tolerance for the long-term survival of skin allograft has a important clinical problem.In recent years, some animal and clinical researches revealed that the donor-specific immune tolerance could be induced in patients who had received hematopoietic stem cells transplantations. However, to induce mixed chimeras, whether with myeloablative and nonmyeloablative preparative regimens, the recipients have to be preparatived with cytotoxic drugs or total body irradiation. After severe burn, the patients show immunosuppressive condition, especially the inhibition of T cell-mediated immunity, making the T cells appear hyporesponsiveness to allogeneic antigen, who are not fit for fierce and injurious preparative regimen. So we should find out a mild one. In particular, the research on MSCs has made a great progress, and many researchers showed that MSCs play a important role in both immunomodulation and hematopoietic reconstitution.This experiment is to induce the donor-specific immune tolerance and prolong the survival time of skin allograft with intra–bone marrow - bone marrow transplantation(IBM-BMT)combined with mesenchymal stem cells (MSCs) co-transplantation.【Purposes】To investigate the possibility to induce the donor-specific immune tolerance and prolong the survival time of skin allograft with intra–bone marrow - bone marrow transplantation(IBM-BMT)combined with mesenchymal stem cells (MSCs) co-transplantation, without myeloablative and nonmyeloablative preparative regimens.【Contents】1. MSCs were isolated from both mice bone marrow and adipose tissue, then cultured in vitro, examined the morphology, proliferation and ability to multipotential differentiation.2. MMC-treated MSCs, which were isolated from both mice bone marrow and adipose tissue, were co-cultured with PHA-stimulated lymphocytes, and then examined the proliferation by MTT method and the expression of foxp3 protein and gene by Western blot and real-time quantitative RT-PCR.3. Mice and rabbits were induced the stable mixed chimeras with intra–bone marrow - bone marrow transplantation(IBM-BMT)combined with mesenchymal stem cells (MSCs) co-transplantation. Then, the formation of tolerance was verified by skin allograft. The skin allograft survival, the formation of chimera and mixed lymphocyte reaction were observed. The expression of foxp3 protein and gene were examined by Western blot and real-time quantitative RT-PCR.【Results】1. MSCs, which were isolated from both mice bone marrow and adipose tissue, were able to be cultured and proliferated in vitro. The primary cells and the passage cells were mostly spindle-shaped, the growth curve showed S-shape. After cells were induced, oil red O stain showed that red lipid droplet existed in cells, cell alkaline phosphatase stain showed that alkaline phosphatase was positive in cells.2. MSCs, which were isolated from both mice bone marrow and adipose tissue, were able to inhabit the proliferation of allogenic T lymphocyte cells. And the foxp3 protein and gene showed obvious high expression.3. The survival time of the skin allograft in the experimental group was obviously prolonged, and the longest one was over 100 days. However, in the rabbit model, there is no obvious difference between experimental group and control group.【Conclusion】1. MSCs are easy to be isolated and cultured in vitro, and proliferated to enough number for pretreatment before BMT.2. MSCs could obvious inhabit the proliferation of allogenic T lymphocyte cells in vitro, and it may be perform through up-regulating the expression of foxp3 in T lymphocyte cells.3. MSCs injection is benefit for the engraftment of hematopoietic stem cells, formation of stable mixed chimeras, immune tolerance, and long-term survival of the skin allograft. However, we did not observe it in rabbit model. The time between bone marrow harvested is so short that the number of hemopoietic stem cells has not yet resumed.