| 1.IntroductionNew potent immunosuppressive drugs have significantly decreased acuterejection, improved short-term effects of renal allograft, but without improvement of recipient/allograft survival. What's more, these drugs are associated with many severe side effects, including nephrotoxicity, susceptibility to opportunistic infections, accelerated atherosclerosis, and malignancy. As a result, the "induction" of donor specific tolerance has been one of the primary goals of researchers in the field of transplantation immunology.Until now, several strategies were used to induce donor specific tolerance, but the realizable way to induce at least some forms of operational tolerance is donor-specific bone marrow (DBMC) infusion. However, the mechanism of DBMC infusion is not clear, in 1993, Starzl proposed that organ allograft acceptance was associated with the survival of bone marrow-derived stem cells, which was assimilated into the larger immunological network of the host, lots of experts consider it's associated with augmentation and maintenance of chimerism. Under such circumstance, recipients can benefit from low dosage ofimmunosuppressive agents, less adverse effects, longer survival of allografts.Until recently, a series of studies in Miami demonstrated that DBMC infusion can significantly increase recipient and allograft survival, decrease acute/chronic rejection, they found that chimerism plays crucial role in inducing and maintaining hyporesponsiveness or unresponsiveness. In our country, one centers showed that chimerism exists in transplant recipients, and frequency of chimerism increaseî–½ith time.In order to explore the regulatory mechanisms involved and to follow the clinical outcome in the hope of eventually reducing the burden of generalized immunosuppression, a series of kidney transplant recipients, post-operatively infused with DBMC in the absence of a bone marrow ablation protocol, have been followed at our center since January 1999. The present study describes the 3-year actuarial patient and graft survival of a series of 63 first cadaver kidney transplant recipients (DBMC group) who received transplants between January 1999 and December 2001, and were given donor vertebral body bone marrow and prospectively followed. Comparing with 425 renal allografts at the same time interval, they were not given marrow treated with an equivalentimmunosuppressive regiment, and followed prospectively. We found DBMCi/'fcuW' infusion is safe and augmention of chimerism can be seen.The objective of this study is to establish PCR-Flow assay to quantitate microchimerism, and try to affirm the effect of DBMC infusion to augment the level of microchimerism, to confirm the correlation between DBMC transfusion and acute rejection, and so on.2.Methods2.1 Experiment partAccording to the accepting and eliminating standards, 77 recipients werescreening out, 36 of them accepted DBMC infusion after cadaver renaltransplantation (DBMC group). 26 of them belong to the control group, and 15of them belong to the long-term survival group who live with the good functional allograft more than 5 years.Peripheral blood mononuclear cells (PBMC) were obtained using Ficoll-Hypaque density gradient centrifugation on schedule (1 week, 4weeks, Sweeks, 22-26weeks, 44-52weeks, over 52weeks for DBMC and control group postoperatively). Then through the extract of genomic DNA, affirmance of the donor and recipients HLA-DR alleles, PCR-Flow assay to detect fluoresceinated peripheral blood mononuclear cells (chimerism or microchimerism).2.2 Clinical partFrom January 1999 to December 2001, excluding multi-organs transplant, second transplant, early death (within one month), delayed graft function, there were 63 patients (observed group) who infused with donor vertebral body bone marrow cells for three times postoperative!}, 406 patients (control group) who were not infused with DBMC at the same interval. Immunosuppression consisted of prednisolone + mycophenolate...
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