| Background: Large area of healthy tissue are required for large size defects repair,autologous tissue often cannot make up for such defects.Allogeneic composite tissue transplantation makes it possible to solve this problem.However,allogeneic composite tissue transplantation faces some challenges,the most critical of which is how to induce immune tolerance to allogeneic tissue and reduce the toxicity or side effects of immunosuppressive regimen.The key to immune tolerance is to make the recipient immune system not attack donor-derived tissue.Allogeneic hematopoietic stem cell transplantation or bone marrow transplantation has been recognized as an important method of inducing immune tolerance.Allogeneic bone marrow cells have donor-derived hematopoietic stem cells,which could engraft in the recipient bone marrow cavity and mature into functional immune and antigen presenting cells.These allogeneic cells do not attack the donor-derived cells and tissue,thereby extending the survival of allografts.Allogeneic bone marrow transplantation still has two problems.First,allogeneic bone marrow transplantation has the potential to change the recipient immune system,but allogeneic bone marrow cells themselves will still induce the rejection from recipient immune system.So allogeneic bone marrow cells still require large doses of immunosuppression or irradiation which lead to unavoidable side effects.Second,just as not only the seeds but also the soil are required for a plant,allogeneic hematopoietic stem cells require donor-derived bone marrow microenvironment for successful engraftment.However,conventional intravenous methods have encountered bottlenecks in solving these problems.It is reported that the efficiency of hematopoietic cell engraftment after intravenous injection of bone marrow cells is relatively low,and the efficiency of allogeneic hematopoietic stem cells engraftment can be improved by intra-bone marrow transplantation.Furthermore,it is difficult to substitute stromal cells and bone marrow cells in allogeneic bone marrow simultaneously by the intravenous approach.Although the possible reasons for better induction of allogeneic hematopoietic stem cell engraftment than intravenous injection were explored by some scholars,the changes in microenvironment in local bone marrow and the changes of allogeneic hematopoietic stem cell function in allogeneic bone marrow transplantation remain unexplored.Immune regulation in local stromal environment lacks further exploration and there are still few studies on the changes of local bone marrow microenvironment and the changes of cytokines associated with immune regulation.Objectives: 1)To explore whether the intra-bone bone marrow transplantation can induce the long-term survival of skin allografts;2)To explore whether the method of intra-bone bone marrow transplantation can induce the changes of local stromal and immune microenvironment,thus prolong survival of skin allografts and hematopoietic system reconstruction;3)To explore whether intra-bone bone marrow transplantation combined with local irradiation can extend the long-term survival of skin allografts and reduce the side effects caused by total body irradiation.Methods: 1)Establish the model of immune preconditioning and allogeneic skin graft transplantation: Recipient mice receives one dose of total body irradiation(4 Gy X ray)combined with intra-bone marrow injection of 5×106 donor-derived bone marrow cells.3 weeks after the bone marrow transplantation,all recipient mice received donor-derived skin grafts with a size of 1 x 1cm on the lateral thoracic region.When the skin graft was rejected,the skin graft was harvested for HE staining.2)Assessment of peripheral blood chimerism: 1 week,3 weeks,12 weeks and 18 weeks after bone marrow transplantation,peripheral blood was collected from the retro-orbital venous plexus of the recipient mice.The flow cytometry was used to measure donor-derived cell percentage in the total peripheral blood leukocytes,granulocytes,T cells,B cells.3)The change of recipient bone marrow microenvironment: The percentage of donor-derived hematopoietic stem cells(H-2Kb + c-kit + sca-1 + lineage-),stromal cells(H-2Kb + CD45-CD31-TER119-),stromal stem cells(H-2Kb + CD45-CD31-TER119-PDGRR-? +),and regulatory T cells(CD4 + Foxp3 +)in the bone marrow(injected or non-injected bone marrow)and spleen were assessed at 3 weeks and 12 weeks after the bone marrow transplantation.4)Immune regulation change in recipient bone marrow and spleen: The expression of IL-2,IL-10 and TGF-? in bone marrow and spleen were measured by real-time quantitative PCR and Western Blotting respectively at 3 and 12 weeks after bone marrow transplantation.5)Surgical removal of local injected bone marrow: After bone marrow transplantation,injected bone was removed at 7 day and 21 days.3 weeks and 12 weeks after surgery,the chimerism in peripheral blood and spleen was measured.Allogeneic mice skin grafts were used to detect whether immune tolerance was established.6)Local irradiation model: A 3 mm-thick lead plate was customized and the left tibia was exposed to 30 Gy X-ray irradiation.After local irradiation,the effect of local irradiation was observed through pathological sections and cell counting in the irradiated or non-irradiated bone marrow.The weight was measured as an indicator of the health of recipient mice after irradiation.7)Statistical Analysis: Student t test was used to compare data from intravenous group,intra-bone bone marrow transplantation,sham group.Paired t test was used to compare data from injected and the non-injected bone marrow in recipient mice.Log-rank test method was used to analyze the data of skin allograft survival.P value less than 0.05 was considered statistically significant.Results: 1)Intra-bone bone marrow transplantation could prolong the survival of skin allografts compared with intravenous injection of bone marrow cells and induce higher chimerism level in peripheral blood.Compared with intravenous injection and sham operation group,intra-bone bone marrow transplantation could extend the survival of skin allografts and reduce the extent of skin graft rejection(Median Survival Time:IBBMT,60 days;Sham Group,32.5 days;Intravenous group,29.5 days).Higher percentage of donor-derived cells were found for the intra-bone bone marrow transplantation group,especially in early peripheral blood donor-derived granulocyte levels.2)Intra-bone bone marrow transplantation of bone marrow cells mainly changed the local stromal cell microenvironment of the injected bone marrow.Compared with the intravenous and sham-operated groups,stromal cells in the injected bone marrow could be replaced by donor-derived cells with higher percentage of donor-derived stromal stem cells at 3 and 12 weeks after bone marrow transplantation(12 weeks after BMT,average 76.2% and 33.3% donor derived stromal cells in injected and non-injected bone marrow respectively).Meanwhile,there were higher level of hematopoietic stem cell and PDGFR-? positive stromal stem cells in the injected bone marrow.3)Intra-bone marrow injection of bone marrow cells can increase recipient-derived regulatory T cells in the injected bone marrow.In contrast to uninjected bone marrow,intra-bone bone marrow transplantation of donor derived cells can increase the proportion of recipient-derived regulatory T cells in the bone marrow at the early stage(3 weeks),and this difference gradually disappeared in later stages(21 weeks).Meanwhile,the increase in the proportion of regulatory T cells was also associated with IL-10 and other cytokines increase,but TGF-? did not increase.4)Local irradiation combined with intra-bone bone marrow transplantation and rapamycin could increase donor-derived cells but not prolong the survival of skin allografts.Local irradiation combined with rapamycin and intra-bone bone marrow transplantation can increase the proportion of donor cells in peripheral blood but not enough to induce immune tolerance or extend the survival time of skin allografts compared with intravenous control.This method reduce the side effects caused by total body irradiation.5)Local trauma by intra-bone bone marrow transplantation was not enough to change the local bone marrow microenvironment.In all of the above experiments,local injection of PBS did not result in a significant change of donor cells,hematopoietic stem cells,stromal cells,stromal stem cells,regulatory T cells between IBBMT and SHAM group.Conclusions: In our studies we have findings below: 1)In this study,we established an animal model of skin allograft transplantation induced by intra-bone bone marrow transplantation of donor derived cells and non-lethal preconditioning.Furthermore,we confirmed that intra-bone bone marrow transplantation could prolong survival of skin allografts compared with intravenous approach.2)This study demonstrated for the first time that intra-bone bone marrow transplantation can replace the stromal microenvironment of locally injected bone marrow and elevate the expression of local regulatory T cells and immunosuppressive cytokines such as IL-10.3)By the removal of injected bone marrow,we found that the injected bone marrow play a critical role in an early stage(7 days)for intra-bone bone marrow transplantation.However,this effect was limited in change the chimersim level and survival time of skin allograft.4)For the first time we combined local irradiation and intra-bone bone marrow transplantation and found that a microchimerism can be induced,this combination was still insufficient to prolong the survival of skin allografts compared with the conventional intravenous method.5)Inflammatory stress caused by intra-bone bone marrow transplantation can't account for the fact that intra-bone bone marrow transplantation could induce longer survival of skin allograft and modify the bone marrow microenvironment compared with intravenous group. |
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