【作者】 李鹏；

【导师】 刘永锋；

【作者基本信息】 中国医科大学 ， 外科学， 2009， 硕士

【摘要】 目的胰岛移植虽然发展前景良好,但仍面临着需要供胰量大,存活率较低,自身免疫性疾病复发所致移植物失功等问题。在临床经门静脉胰岛移植中,移植后大量胰岛丢失是主要的问题之一。在移植过程中,注入门静脉的胰岛与血液接触,发生一种凝血/炎症反应过程,我们称之为立即经血液介导的炎症反应(IBMIR)。IBMIR以凝血和补体系统的快速激活,胰岛内白细胞的募集和渗入,血小板的快速粘合和激活为特征。组织因子是IBMIR的主要启动因子。本实验旨在观察经胰岛—内皮细胞复合体的方法预处理的胰岛对IBMIR的影响,为临床胰岛移植胰岛预处理提供一种有效的新方法。材料与方法1、实验动物及分组本实验采用雄性Wistar大鼠(中国医科大学实验动物中心提供),体重250～300g。实验分为三组:一组将分离、纯化的胰岛和大鼠主动脉内皮细胞混合培养,形成胰岛—内皮细胞复合体,称为实验组;一组将分离、纯化的胰岛直接置于CMRL1066培养液中培养,称为对照组;一组为PBS液,称为阴性对照组。2、胰岛、内皮细胞的获取,胰岛—内皮细胞复合体的制备,胰岛功能的测定和模拟体内循环模型的建立大鼠胰腺原位灌注消化液、不连续密度梯度离心法获得纯化胰岛。胶原酶消化贴片法获取大鼠主动脉内皮细胞,并传代培养。胰岛、内皮细胞混合培养5天,获得内皮细胞包被的胰岛,即胰岛—内皮细胞复合体。培养过程中进行葡萄糖刺激试验以测定胰岛素释放指数。实验组、对照组及PBS阴性对照组分别置于模拟体内循环模型的3个管中反应,分别于灌注前,5分钟、15分钟、30分钟、60分钟留取血浆进行分析。3、观察指标对不同时间阴性对照组、对照组和实验组血浆中的TAT、C3a、血小板的数量进行测量和统计。通过HE染色和免疫组化染色进行形态学观察。4、统计学分析将所得数据用均值士标准差表示,采取t检验和方差分析,P＜0.05认为有统计学意义,所有数据用SPSS11.5软件处理。结果实验组和对照组血浆中的TAT、C3a、血小板的数量在灌注后60分钟有明显差别(P＜0.05)。免疫组化染色提示胰岛—内皮细胞复合体组较未包被组中性粒细胞侵润明显减少。结论1、胰岛—内皮细胞复合体并不影响胰岛的功能。2、胰岛—内皮细胞复合体能够显著减少胰岛移植初期的凝血激活、补体激活、血小板消耗和炎细胞浸润,从而显著减轻IBMIR的反应。3、胰岛—内皮细胞复合体的方法有可能成为临床胰岛移植中胰岛预处理的一种新方法。更多还原

【Abstract】 PrefaceAlthough islet transplantation are good prospects for development,it is still faced with the need for pancreatic mass,a lower survival rate,recurrence of autoimmune diseases caused by graft failure and so on.In clinical islet transplantation through portal vein,the substantial islet loss after transplantation is one of the main problem.At transplantation,the portal vein injection of insulin and blood contact,the occurrence of a coagulation/inflammatory response process,we call the instant blood mediated inflammatory reaction.(IBMIR) IBMIR are characterized with coagulation and complement system activation,the collection and infiltration of leukocytes and platelets adhesion and activation.Tissue factor is the main initiation factor IBMIR.This experiment is to observe effect of composite islet-endothelial cell grafts to instant blood mediated inflammatory reaction in islet transplantation in rat.Materials and MethodsAnimal:SD rat of either sex with weigh ranging from 250-300g as the donor, provided by animal center of China medical university.Purified islets are obtain by perfusion of rat pancreatic digestive juice in situ and discontinuous density gradient centrifugation.The endothelial cells are obtained by collagenase digestion method from patch rat aortic,and cultured randomly divided into 8 groups Islet and endothelial cells co-cultured 5 days,accessing to islet coated endothelial cells,that is,composite islet-endothelial cell grafts.In the process of cultivate,we obtain insulin release index with glucose-stimulated test.Coated group,uncoated group and PBS control group were placed in simulated blood circulation system in the reaction tube 3,respectively, before reperfusion,5 minutes,15 minutes,30 minutes,60 minutes of collecting plasma for analysis.TAT,C3a and platelet are measured.Through HE staining and immunohistochemical staining we have a morphological observation.Statistical analysis:all the data expressed in meanlSD.The significant of difference was evaluated by t test or ANOVA.P＜0.05 was considered having statistical significance.ResultsThe plasma TAT,C3a and the number of platelets in uncoated group and the composite islet-endothelial cell grafts group at 60 minutes after reperfusion have a significant difference(P＜0.05).The neutrophil filtration decreased significantly in uncoatedgroup than in composite islet-endothelial cell grafts group from immunohistochemical staining of pancreatic islet tips.Conclusions1.The composite islet-endothelial cell grafts does not effect the function of islet.2.The composite islet-endothelial cell grafts can significantly reduce the initial islet transplantation activation of coagulation,complement activation,platelet consumption and inflammatory cell infiltration,which significantly reduced the response IBMIR.3.The way of composite islet-endothelial cell grafts provides a new method for clinical pancreatic transplantation.更多还原