| Part 1 The in vitro research of the immunologic modulatory effects of human bone marrow stem cellsInduction of donor-specific tolerance has been a long sought after goal in human organ transplantation. There was little breakthrough in clinic tolerance induction in despite of the successful induction of tolerance animal models. The "microchimerism theory" brought forward by Strazl promoted the clinic tolerance induction for improving the long term survival of organ allografts by donor bone marrow cell (DBMC) transfusion. But the mechanism of tolerance induction by DBMC is not clearly understood, and the DBMC infusion accompanying organ transplantation may cause a number of possible conflicting immunological effects. Aside from possibly immunizing the recipient against the donor, (1)the infused bone marrow cells may respond to the recipients, (2)subpopulations of cells in the DBMC may regulate the alloreactivity of the infused donor cells to the recipients, and (3) the infused bone marrow cells may down-regulate responses of the recipient to the donor alloantigens. Mathew, et al, documented a series of in vitro culture experiments to investigate the effects of DBMC in tolerance induction. He comfirmed that: Firstly, The DBMC had the regulatory role in immune responses against donor cell alloantigens and the modulator/ effect reguired the direct cell-cell contact and living cells; Secondly, DBMC could proliferate as responding cells in co-culture with x-irradiated allogeneic cells and could also cause regulatory effects when added as a third component to MLC reactions, their responding effects can been prevented by low dose immunosuppression agents, but the common used immunosuppression agents have no influence their regulatory effect; Thirdly, The DBMCs possess immunogenicity but clearly weaker than donor allogeneic spleen cells. These results indicate that DBMC are able to function as effective in vitro stimulators, and that the immune responses mediated by them can be down-regulated by their own inherent suppressive nature, an effect that can be enhanced by the presence of immunosuppressive drugs. There were little research about the effect of multiple subpopulations in DBMC, especially, the effect of CD34 positive stem cells. The CD34 positive stem cells possess the ability of self replication and multiple differentiation potential, The chimerism cells of donor's found in combined DBMC infusion organ transplant recipients maybe totally differentiate from CD34 positive stem cells origin. The characteristic of high expression of HLA DR molecular whereas low expression of B7-1 and B7-2 Costimulation moleculars on the surface of CD34 positive cells made them "nature tolerance induction cells". Here, We researched the directly immunologic modulatory effects of CD34 positive cells in DBMC, and also investigated their effective dosage, whether donor specific and the effect of common used immunosuppressive agents.Materials and methods1. The separation of the cells for in invtro cultureHarvest the mononuclear cells from the spleen and vertebral Body of cadaveric donor.2. The positive isolation of CD34 positive stem cellsIsolation the CD34 positive stem cells by MACS cell isolation systems and Direct CD34 progenitor cell Isolation Kit. And analysis their surface molecularsby flow cytometer.3. The immunological regulatory effect of donor bone marrow origin cells on mixed lymphocytes reaction(MLR)The blood group matched and HLA DR unmatched cadaveric donor spleen cells were served as responsing cells and stimulating cells respectively, The bone marrow mononuclear cells, CD34 positive cells and CD34 negative cells were served as modulatory cells respective. In a in vitro MLR system, theregulatory effect and whether donor specific were observed.4. The effect of multiple combination of immunosuppression agents on the modulatory of CD34 positive cellsTo observation the clinic common used combination of immunosuppres... |
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