The Research Of Draining Lymph Nodes On Composite Tissue Allotransplantation

The emergance of new immunosuppressive drugs leads to a big success in clinical organ transplantation. In addition, the success pushes the composite tissue allotransplantation to be a reality. However, life-long dependence on the immunosuppressive drugs will increase the incidence of side effects of immunosuppression. Without better immunosuppressive drugs, we need to try the best to decrease the dose of system immunosuppression. The composite tissue allgrafts are usually superficial for investing and interfering. As a result, local immunosuppression may be a better way to decrease the system immunosuppression.T lymphocytes are the principle component of adaptive alloimmune response. Na?ve T cells within the blood could enter secondary lymphoid tissues via HEV and contact with APCs, which present the alloantigens. Then na?ve T cells start to mature to effector T cells, which will get back to the blood and get to the allografts finally, and start the rejection to the allografts. The high immunogenecity of skin in CTAs is the main reason for leaving behind by organ transplantation. Therefore, conquering the rejection of skin is the key to support the development of CTA. The antigen of skin allografts digested by APCs only went through lymph to draining lymph nodes (dLNs). Therefore, the method of interrupting the dLNs efficiently will significantly decrease the effector T cells, and lower the rejection response. This will decrease the system dose of immunosuppression. The research aimed to the effect of dLNs on the compotise tissue allografts.Experiment 1 Anatomy of rat lymph nodes and selection of best allotransplant rat modelObjective To locate the draining lymph nodes of various skin flap of rats by anatomy of whole lymph nodes of rats and to choose a best animal transplant model for the research of lymph nodes by the different characteristics of various draining lymph nodes. Methods SD and Lewis rats will be anatomized for the whole body lymph nodes. The location, shape, amount, weight and cell consentration of lymph nodes will be record and analysis. The difference of lymph nodes between two strains of rats or two different weights will be compared. Injection of blue ink will be performed subcutaneously in various places in rats for locating the draining lymph nodes. The best animal transplant model will be chosen based on the draining lymph nodes and easiness of accessible flaps. Results The locations of the lymph nodes of two strain rats are similar. The amount and weight of lymph nodes are stable. The lymph nodes weights of SD rat are heavier than those of Lewis rat. The heavier the rat is, the heavier the lymph nodes. Epigastric flap is selected to be the best model among three good flaps for the research. And the epigastric flap transplant model is established successfully. Conclusion The draining lymph nodes of epigastric flap are contralateral inguinal LNs, ipsilateral internal iliac LNs, and ipsilateral axillary LNs. Moreover, the epigastric flap transplant model is the best for the research of draining lymph nodes.Experiment 2 The effect of rapamycin intraperitoneal injection on allografts of ratsObjective To make sure the lowest and efficient immunosuppressive dose of rapamycin on the survival of epigastric flap allografts by rapamycin intraperitoneal injection of different doses. Methods BN and Lewis rats were applied as donor and receiptor, respectively, in epigastric flap allotransplantation. The experiment was divided to five groups according to the various rapamycin doses: 8, 4, 2, 1, 0mg/kg/day. The duration of the drug application is from day 1 of pre-operation to day 7 of post-operation. The mean survival time, the quality of the allografts, pathology will be invested. Results The completely animal sample number was 36, and useful animal sample number was 29. The mean survival time of the groups are 17.3±1.7 days, 17.5±1.7 days, 14.2±1.3 days, 7.3±0.6 days, 7.3±0.5 days, respectively. Conclusion the lowest and efficient immunosuppressive dose of rapamycin on the survival of epigastric flap allografts is 2mg/kg/day.Experiment 3 The effect of removing draining lymph nodes on the survival of rat allograftsObjective To study the effect of removing draining lymph nodes on the survival of allografts. Methods BN and Lewis rats were applied as donor and receiptor, respectively, in epigastric flap allotransplantation. The experiment was divided into five groups randomly. Group A: laparotomy 2 weeks before transplant control; Group B: laparotomy 2 weeks before transplant and rapamycin 2mg/kg/day for 9 days (-1~7); Group C: removal of draining lymph nodes 2 weeks before transplant; Group D: removal of draining lymph nodes and spleen 2 weeks before transplant; Group E: removal of draining lymph nodes 2 weeks before transplant and rapamycin 2mg/kg/day for 9 days (-1~7). The mean survival time, IL-2 levels of serum are investigated for the effect of removing draining lymph nodes. The effect of removing draining lymph nodes in different time is also investigated. Results The mean survival time of the five groups are 7.3±0.5 days, 14.4±0.3 days, 9.5±1.0 days, 8.8±0.8 days, 17.0±1.9 days. The IL-2 levels of Group B, E are significantly lower than those of Group A, C and D. The IL-2 levels of Group C, D are slightly higher than Group A. The mean survival time among different times of removing draining lymph nodes are similar. Conclusion Removal of draining lymph nodes could prolong the mean survival time of rat allografts and with low dose of rapamycin could prolong the MST of allgrafts.Experiment 4 The machnism study of lymph nodes and lymphatic drainage in transplant immunityObjective To study the machnism of lymph nodes and lymphatic drainage in transplant immunity by investigate the response of lymph nodes to alltransplant and the effect on allografts survival time after separate the skin contact between host and graft. Methods BN and Lewis rats were applied as donor and receiptor, respectively, in epigastric flap allotransplantation. Lymph nodes are collected 1, 3, 5, 7, 9 days respectively after transplant. The control is designed by Lewis self-epigastric flap removal without cutting off the vascular pedicle and sewing back after a while. The weight, cell concentration, CD4+CD25+ cells were investigated for dynamic analysis. The flap tube allotransplant model between SD and SD rat or BN and Lewis rat with or without connection between host and graft skin will be established. The survival time and quality of the flap tube will be judged. Results The draining lymph nodes after transplant were congestive, enlarged, and increased by weight, CD4+CD25+cells also increased compared with non-draining lymph nodes. The indicators of inflammation control are similar to the transplant group except the slower increase of LN weight. The longest survival time is 32 days in flap tube allotransplantation model without connection between host and graft skin. The other three groups all failed to prolong the survival time of allografts. Conclusion Draining lymph nodes are the principle response place after alltransplantation. Separation of skin connection between host and grafts could prolong the survival time of allografts only across a weak MHC barrier between donor and receiptor.