| It has been nearly half of a century since Dr. Barnard performed the first human heart transplantation in 1967, and there was a great development in heart transplantation during this period. Now heart transplantation is considered as the most effective therapy in treatment of end-stage heart diseases. However, rejection of allografts is still a tough problem that troubles cardiac surgeons. Recently, granulocyte colony-stimulating factor (G-CSF) which is an important hematopoietic growth factor of the myeloid lineage was shown a profound immunoregulatory effects in adoptive immunity and T cell tolerance. But the mechanism is still not very clear. The study objective is to investigate the potential mechanism of G-CSF's anti-rejection effect in a rat fully mismatched cardiac allograft model.Objective: To explore the surgical skills and modify the procedure in making animal models of heterotopic heart transplantation in rats, in order to make it more convenient and improve the success rate. And to investigate the potential mechanism of G-CSF's anti-rejection effect in this modified animal model in order to provide a possible theory for the heart transplantation therapy and other autoimmune diseases.Methods: 1. Heterotopic heart transplantations were performed by using Ono's method with modifications of preparation of recipient, donor cardiectomy and vascular anastomosis. 2. Four groups received the allograft by subcutaneous injection with rh-G-CSF at a dose of 0μg/kg/day, 125μg/kg/day, 250μg/kg/day, and 500μg/kg/day respectively for six days starting the day of cardiac transplantation. Observations included: allograft histological examination, mixed lymphocyte reaction (MLR), and allograft lifespan. 3. Cytokine and cellular profiles were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Regulatory T cells'presence and suppressive function were determined by adoptive cells transfer experiments.Results: 1. Of the 100 cases receiving heterotopic abdominal heart transplantation, complete success was achieved in 89 cases. Total ischemia time was (32±5)min, and full time of operation was (60±10)min. 2. The rats in the 250μg group and 500μg group showed significant alloreactive T-cells hyporesponsiveness both in vivo and in vitro. However, the data of the 125μg group was not significantly changed as compared with the control group. 3. The T-cells hyporesponsiveness was associated with the enhancement of CD4+CD25+T cells in peripheral blood, high levels of interleukin 10(IL-10) and transforming growth factorβ1 (TGF-?1) production in MLR. The six-day course of rh-G-CSF administration significantly decreased the CD4+CD25+T cells percentages in the bone marrow, and the splenocytes from allograft recipients treated with rh-G-CSF were capable of prolonging secondary heart allograft survival.Conlusions: 1. This improved Ono's method can reduce the ischemia time, make the anastomosis easier and increase the success rate of heterotopic heart transplantation. The whole process can be completed by a single man without microscope assistance, so this method is easy to learn and perform. 2. The immunoregulatory properties of rh-G-CSF are critical in the early control of allograft rejection in rat heterotopic heart transplantation models, and posttransplant treatment of cardiac allograft recipients with rh-G-CSF leads to alloreactive T cells hyporesponsiveness in vivo and in vitro. The anti-rejection effect is relevant to its dosage. 3. Rh-G-CSF can enhance CD4+CD25+ T-cells in peripheral blood and may be the main mechanism of its anti-rejection function. Moreover, IL-10 and TGF-?1 are important effector cytokines in this study.
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