【作者】 李杨；

【导师】 郭树忠； 夏炜；

【作者基本信息】 第四军医大学 ， 外科学， 2010， 博士

【摘要】 近年来,随着免疫抑制药物的不断发展和联合免疫抑制方案的不断进步,临床器官移植取得了巨大的成功,含有皮肤的同种异体复合组织移植(CTA)也进行了初步的尝试,并取得良好的效果。但是,终身使用免疫抑制药物带来的各种毒副作用延缓了CTA的进一步发展。在更好的免疫抑制药物出现之前,如何降低现有免疫抑制药物的全身使用剂量成为研究的热点。而带有皮肤的CTA多位于体表,易于观察和干预,因此,有效的局部免疫抑制有望降低全身免疫抑制用药量。移植免疫反应是以T细胞适应性免疫应答为主。血循环中的初始T细胞在次级淋巴组织与载有抗原的抗原提呈细胞(APCs)经过反复接触、识别,初始T细胞逐渐成熟,并增殖成为效应T细胞,再次进入血液循环,到达异体移植物局部后,促使移植物发生排斥反应。CTA的发展之所以滞后于器官移植,是因为皮肤的强免疫源性,因此解决了皮肤的移植排斥反应将推动CTA的发展。而皮肤移植物的异体抗原被APCs摄取后,只能沿着淋巴引流进入引流淋巴结,很难有大量的抗原通过血液进入脾或其他部位的淋巴结。因此,如果能够有效的干预引流淋巴结,就可能显著减少效应T细胞的产生,减轻移植物的排斥反应,进而降低免疫抑制药物的全身用药量,减少毒副作用。因此,本研究拟针对引流淋巴结在CTA中的作用进行研究。实验一大鼠淋巴结解剖及异体移植模型的选择目的:通过大鼠全身淋巴结的解剖,各部位引流淋巴结的确定,对比研究各种移植模型的引流淋巴结,为淋巴结干预研究选择适当的移植模型。方法:对SD封闭群、Lewis近交系大鼠分别进行全身淋巴结解剖,记录淋巴结的全身分布位置、形态特征、数量、重量及单位重量细胞浓度,比较不同品系间、不同体重大鼠间淋巴结重量、单位重量细胞浓度的差异;大鼠备选皮瓣局部皮下注射美兰,解剖大鼠,定位染色的引流淋巴结;通过分析比较不同移植模型的解剖及引流淋巴结确定本实验移植模型。结果:2个品系大鼠淋巴结的分布基本明确,无明显变异,数量、重量恒定,SD封闭群大鼠淋巴结较Lewis大鼠略重,淋巴结重量随大鼠体重的增加而增加。经颜面部、侧胸、下腹部皮瓣引流淋巴结的确定及三种皮瓣手术难度的评估,确定以下腹部皮瓣为最佳,选定为本研究移植模型。并成功建立下腹部游离皮瓣移植模型,并通过术后淋巴结的解剖验证了下腹部游离皮瓣的引流淋巴结的部位。结论:下腹部皮瓣的引流淋巴结为对侧腹股沟淋巴结、同侧腋固有淋巴结、同侧髂内淋巴结,该皮瓣移植模型是淋巴结干预实验的最佳移植模型。实验二雷帕霉素腹腔注射对大鼠异体皮瓣移植的作用目的:通过雷帕霉素腹部注射对大鼠腹部皮瓣存活的影响研究确定雷帕霉素的最低有效免疫抑制剂量。方法:以BN大鼠为供体、Lewis大鼠为受体,进行下腹部游离皮瓣异体移植;将雷帕霉素原料药配置成1mg/mL的雷帕霉素腹腔注射剂,根据雷帕霉素使用剂量8、4、2、1、0mg/kg/day分为A、B、C、D、E 5组,用药时间-1—7天;观察各组皮瓣存活时间、存活质量、病理表现,确定雷帕霉素最低有效免疫抑制剂量。结果:总实验例数36例,有效例数29例。各组大鼠平均存活时间分别为17.3±1.7天(A组)、17.5±1.7天(B组)、14.2±1.3天(C组)、7.3±0.6天(D组)、7.3±0.5天(E组)。结论:选择2mg/kg/day雷帕霉素剂量作为最低有效免疫抑制剂量。实验三去除引流淋巴结对大鼠异体皮瓣存活的影响目的:通过去除大鼠下腹部皮瓣的引流淋巴结,确定其对皮瓣存活的影响。方法:BN大鼠为供体、Lewis大鼠为受体,将大鼠随机分为5组,A组:移植术前2周开腹假手术对照;B组:移植术前2周开腹假手术,雷帕霉素2mg/kg/day全身用药(-1 ~ 7天);C组:移植术前2周去除引流淋巴结;D组:移植术前2周去除引流淋巴结及脾;E组:移植术前2周去除引流淋巴结,雷帕霉素2mg/kg/day全身用药(-1 ~ 7天)。术后通过观察动物存活率、皮瓣平均存活时间、血清IL-2水平等指标评价去除引流淋巴结对皮瓣存活的影响。对移植前2周,移植同时、移植后3天不同时间去除引流淋巴结对皮瓣存活时间的影响也进行了研究。结果:各组的平均存活时间分别为7.3±0.5天(A组),14.4±0.3天(B组),9.5±1.0天(C组),8.8±0.8天(D组)、17.0±1.9天(E组)。B、E组血清IL-2水平明显低于A、C、D组;C、D组的IL-2水平比A组略高。术前14天、术中、术后3天不同时间去除引流淋巴结,移植后皮瓣存活天数无明显差异。结论:去除引流淋巴结可以延长皮瓣平均存活时间,结合低剂量雷帕霉素免疫抑制可以延长皮瓣存活时间。实验四淋巴结、淋巴引流在移植免疫中的机制研究目的:通过研究淋巴结对异体移植物的反应、阻断淋巴引流对移植物存活的影响,探讨淋巴结、淋巴引流在移植免疫中的机制。方法:BN为供体、Lewis为受体,下腹部异体皮瓣移植术后1、3、5、7、9天不同时间解剖淋巴结,同Lewis自体分离腹部皮瓣保留血管蒂再缝合回去作为对照,对两组大鼠不同时间摘取的引流淋巴结和非引流淋巴结进行称重、细胞计数、流式细胞分析CD4+CD25+细胞的变化,分析引流淋巴结在异体移植后的动态变化。对去除引流淋巴结的Lewis大鼠进行BN下腹部游离皮瓣移植后,获取非引流淋巴结,观察其大体变化、重量变化、细胞计数、流式细胞分析来判断非引流淋巴结是否受异体移植物的影响。建立SD→SD、BN→Lewis的隔离皮肤或不隔离皮肤的皮管移植模型,通过阻断皮肤淋巴引流观察皮管存活时间及质量。结果:移植后的引流淋巴结逐渐充血、肿大、重量增加,细胞密度增高,流式细胞分析发现CD4+CD25+细胞较非引流淋巴结增多。异体移植及单纯炎症均可导致引流淋巴结重量增加、单位重量细胞浓度增加,但移植导致淋巴结重量的增加高于炎症所致,单位重量细胞浓度无明显差别,但较两组的分引流淋巴结均有明显差别。去除引流淋巴结后移植术后,非引流淋巴结未发现明显充血及肿大。隔绝皮肤淋巴引流的SD→SD皮管移植物最长存活至32天,未隔绝供、受体皮肤接触的SD→SD及两组BN→Lewis的皮管移植均未能长期存活。结论:引流淋巴结是同种异体移植后主要反应部位,隔绝皮肤淋巴引流供、受体间在较小的MHC差异下可明显延长异体血管化皮肤的存活时间。更多还原

【Abstract】 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.更多还原