| Introduction Currently the clinical application of organs and cells transplantation was so wide that it has cured some difficult diseases. For example, hematopoietic stem cells transplantation is the most effective therapy measurement for extremely severe acute radiation sickness, but it often occurs graft rejective reaction. Therefore, large dose of radiotherapy and chemotherapy are performed before transplantation, and immunosuppressive agent is used for long term after transplantation. It not only brings big damage to the patients and affects therapeutic effect, but also the graft can't survive for long term. So It becomes an urgent problem to find an effective therapy by which immunosuppressive agent isn't used and the body can prevent acute rejection and chronic rejection and produce permanent endurance for the graft. At the present, it is the best method to solve the above problem to induce the occurrence of specific immunotolerance of donors, which is the hot in research.The research found that the most effective toleragens inducing the immunotolerance were lymphocyte and bone marrow cells, furthermore, donor graft itself is also a necessary toleragen maintaining the tolerance. Bone marrow mesenchymal stem cells possess the ability to multipotency, stable characteristic, weak immunogenicity, few expression of MCH class I molecule, fewer or no expression of MHC class II molecule and T cells costimulatory molecules B7, which have an effect of inhibiting T cells proliferation, so peoples want to use it as toleragen to induce specific immunotolerance. If infusion of MSCs can induce immunotolerance, then transplanted organs and cells as persistent toleragen maintain this immunotolerance state, in addition, MSCs have an effect of migrating to damaged tissue and repairing the injury, and MSCs transplantation not only will produce short-term therapeutic effect but also graft will survive for long term without graft rejective reaction under the condition of using few or no immunosuppressive agents. It will solve the most difficult problem in transplantation field, such as the therapy of extremely severe radiation sickness using bone marrow transplantation.The study want to establish a simple xenogenic liver transplantation animal model, induce the immunotolerance by the infusion of MSCs, and provide a new idea for solving the problem on graft long-term surviving.Objective To explore the possibility and mechanism of induction of the immunotolerance to xenogenic liver transplantation by mouse MSCs, establish a simple, easy animal model for the research of immunotolerance and provide a new way to induce transplantation immunotolerance.Methods1. MSCs were isolated from mice bone marrow, purified and expanded combining the adherent culture with digestion. The morphology and growth characteristic of MSCs were observed and recorded under inverted microscope, and cell growth curve and cell cycle were determined. 2. MSCs were induced to differentiate into osteoblasts in inducing me- dium containingβ-sodium glycerophosphate, dexamethasone, and antiscorbic acid. It was confirmed that MSCs possessed multipotency by alkaline phosphatase staining.3. The expression of CD34 was determined by flow cytometry for identi- fying MSCs.4. The expression of MHC class II molecule was detected by immuno- cytochemistry.5. The migration and residence of MSCs in thymus, spleen, bone marrow and liver transplantation region were observed by laser scanning confocal microscope 24h, 5d and 10d after DAPI-labeling mouse MSCs were transplanted into rat by the vena caudalis.6. The changes of the appearance and tissue of the transplantation region were observed in model rats with xenogenic liver transplantation which were establish by embedding mouse hepatic tissue pieces into the cut of rat liver.7. Experimental animals were randomly divided into 4 groups: normal control group in which the rats were not treated, model group in which liver transplantation was performed without therapy, treatment group I in which mouse hepatic tissue pieces were transplanted into rats 17d after mouse MSCs were injected into rats, and treatment group II in which mouse MSCs were injected into rats 2d after liver transplantation. The injected MSCs density was 1×106cells/ml with the quantity of 0.8ml/200g. The inhibition effect of mouse MSCs on the lymphocyte proliferation of rat thymus and spleen at different time was tested by in vitro lymphocyte proliferation test. The expression of CD4+,CD8+and CD25+ lymphocyte was detected by flow cytometry. The repair of hepatic tissue injury in rats with xenogenic liver transplantation was observed by HE staining.Results1. The purified and steadily passaged MSCs were screened by combining adherent culture with digestion after MSCs were isolated from mouse bone marrow. The result of cell cycle showed that more than 73% MSCs were G1/G0 phrase, and high proportional G1/G0 phrase cells suggested that MSCs possessed multipotency.2. It showed that alkaline phosphatase staining was positive that the dark grey sedimentation appeared in the osteoblasts differentiated from MSCs.3. Mouse MSCs didn't express CD34 by flow cytometry.4. Mouse MSCs didn't express MHC class II molecule by immunocyto- chemistry.5. DAPI-labeling mouse MSCs were found in rat liver transplantation region, immune organs such as thymus, spleen and bone marrow and so on by laser scanning confocal microscope.6. The incision appeared gray, mouse live tissue was enclosed by rat liver tissue which were observed in the tissue section, and abnormal changes occurred in rat livers tissue in killed rats transplanted hepatic tissue pieces.7. The lymphocyte proliferation of rat thymus and spleen was inhibited in mouse MSCs treatment group, and the values in treatment group were significantly lower than those in control group. MSCs changed the phenotype of lymphocyte in peripheral blood. And the number of CD8+ T cells was significantly increased. On the 2th, 5th, 10th, 15th days after therapy using MSCs, the number of infiltrative inflammatory cells decreased and liver tissue structure was observed in transplantation region.Conclusion The mouse MSCs isolated in this study were different from hematopoietic cell lines, which can migrate and reside in immune organs and transplantation region of liver tissue, participate in the repair effect of liver tissue in transplantation region, and can inhibit the proliferation of lymphocyte of rat thymus and spleen in model rats with xenogenic liver transplantation, result in the up-regulation of the levels of CD8+ T cells. The results showed that mouse MSCs could induce rat to produce immunotolerance in model rats with xenogenic liver transplant by the low immunogenicity of MSCs, the inhibitory effect of MSCs on T cell proliferation and regulation of MSCs on T cells subgroup. This study succeeded in establishing a simple and easy animal model for studying immunotolerance, which provide a new route for inducing transplantation immunotolerance.
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