猪到人异种心脏移植超急性排斥反应的研究

【作者】 刘波；

【导师】 詹金彪；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 【目的】随着基础研究的不断深入及外科技术的日益完善，器官移植已成为救治多种器官功能衰竭患者生命的首选方法；然而随着人体器官移植适应症的增加和移植成功率的提高，器官移植手术数量大幅增加，造成临床上器官来源呈现严重短缺。为了解决这个问题，近年来，异种器官作为缓解人体器官短缺的有效措施之一受到了人们的关注。在异种动物之中，由于猪器官的生理解剖特点及脏器大小与人类十分接近，被认为是异种器官移植的理想供体，但猪→人异种器官移植需要克服的首要免疫学障碍是超急性排斥反应。其发生的主要机理是人体内预先存在的天然抗体识别猪细胞表面的异种抗原表位—半乳糖α1，3半乳糖（galactoseα1，3 galsctose，Galα1，3 Gal），从而激活补体而引起血小板聚集，形成血栓，导致移植器官的失功。水苏糖是从一种叫做地灵（Stachys floridona）的根茎中提取的α半乳糖苷类非还原性低聚糖，简单的结构表示为：果糖—葡萄糖—半乳糖—半乳糖（Galα1，6-Galα1，6-Gluβ1，2-Fructose），分子式为C₂₄H₄₂O₂₁，分子量为666.59，无毒，易溶于水。水苏糖具有α-Gal抗原相似的分子结构，理论上具有抑制超急性排斥反应的作用。本实验旨在从细胞及器官水平，观察水苏糖对猪到人异种心脏移植超急性排斥反应的抑制作用。【方法】首先，将传代培养的猪髋动脉内皮细胞PIEC暴露于人血浆中，同时与水苏糖或PBS（空白对照）共同孵育后，采用MTT法检测细胞的存活率，从而观察水苏糖对PIEC的保护作用。为观察对整体器官的效应，我们建立了离体猪心脏体外灌流的超急性排斥反应模型，检测人血液单独灌注组及人血液加水苏糖灌注组心脏组织的病理及其免疫组化（IgG及IgM在组织的沉积）的变化，以观察水苏糖对猪到人异种心脏移植超急性排斥反应的抑制作用。【结果】1．MTT法检测水苏糖处理组的PIEC的相对存活率随着水苏糖浓度的增加而升高。说明水苏糖对PIEC有保护作用。2．成功地建立了人血液体外灌注猪离体心脏的超急性排斥反应的模型。3．灌注猪心脏的存活时间：水苏糖加人血液灌注组的存活时间明显长于人血液单独灌注组。人血液单独灌注猪心脏的平均存活时间为（9.5±2.5）min，而人血液加水苏糖灌注猪心脏的平均存活时间为（46.8±8.1）min。4．灌注猪心脏组织的病理学检查：水苏糖加人血液灌注组的猪心脏未发生超急性排斥反应；而人血液单独灌注组猪的心脏的心肌间质弥漫性出血、水肿，血管扩张，内皮细胞肿胀、坏死，细胞间质散在单核细胞浸润，证明发生了超急性排斥反应。5．灌注猪心脏组织的免疫组化检测：水苏糖加人血液灌注组的猪心脏心肌血管内皮未见IgG及IgM的沉积；而人血液单独灌注组见明显沉积。【结论】上述实验结果说明水苏糖可以与人血液中的天然抗体竞争性结合，具有抑制异种移植超急性排斥反应发生的作用，从而对暴露于人血液中的PIEC及猪心脏有保护作用。【目的】器官移植已经成为许多终术期器官疾患的主要治疗措施之一，但它的广泛应用引起了人体可移植器官的严重短缺，所以人们开始认真地考虑异种移植。目前，猪到人异种移植已经被公认为解决器官短缺的有效措施。但猪到人的异种移植存在很多免疫学障碍。除了猪到人的超急性排斥反应、急性血管性排斥、和慢性排斥反应外，猪和人的血型不相容等因素也可能是引起异种器官移植物失功的原因之一。所以本实验旨在检测猪红细胞上是否表达与人类相似的ABO血型抗原。【方法】1．猪红细胞表面A、B抗原的检测采用细胞凝集法。猪红细胞和抗-A、抗-B抗体在血凝板中反应，红细胞凝集时，成网环状沉积在血凝板底部，说明具有相应抗原；不凝集时形成点状沉淀，说明不表达相应抗原。2．猪血浆中抗-A、抗-B抗体的检测采用血型反定型法检测猪血浆中的血型抗体。将猪的血浆预先与人类O型红细胞孵育，以去除猪血浆中存在抗人抗体引起的非特异型凝集。【结果】1．猪红细胞表面A、B抗原的检测我们检测了72头不同种猪的红细胞，未检测到A或B抗原。2．猪血浆中抗-A、抗-B抗体的检测我们检测了72头猪血浆中的抗-A、抗-B抗体，其中37头猪检测到抗-A和抗-B抗体，14头猪检测到抗-A抗体，3头猪检测到抗-B抗体，18头猪未检测到抗-A或抗-B抗体。【结论】上述实验结果说明在我们检测的72头猪中，红细胞上不表达与人类相似的血型抗原，但猪的血浆中存在血型抗体。我们检测的72头猪的血浆中，51.4％具有抗-A和抗-B抗体，19.4％具有抗-A抗体，4.2％具有抗-B抗体，25％不具有抗-A或抗-B抗体。更多还原

【Abstract】 Objective: Although allotransplantation, with its great basic and clinical success, has become the potential treatment to end-stage organ failure, it also has resulted in a shortage of available organs from deceased human donors. In recent years, attention has been drawn towards xenotransplantation. Pig is investigated as the most likely species to provide organs for human because the anatomy and organ size of pig is similar with human. However, major problems remain to be resolved before successful clinical xenotransplantation can be initiated. The initial immunological barrier of pig-to-human xenotransplantation is hyperacute rejection, which is initiated by natural preformed antibodies directed a carbohydrate epitopes （α-Gal） on the pig vascular endothelium. The binding of antibody to antigen activates the complement cascade, which leads to graft damage.Stachyose is extracted from the root of stachys floridana. It is a kind of nonreductive oligosaccharides. Its constitutional formula is fructose-glucose-galactose-galactose with molecular formula C₂₄H₄₂O₂₁, and molecular weight is 666.59. Stachyose is not toxic and water-soluble. It can inhibit hyperacute rejection of xenotransplantation theoretically since it contains the structure of α-Gal which is similar with the xenogenic antigenic epitope of galactose-α1,3-galacotse. Ourobjective was to investigate the inhibition of stachyose on hyperacute rejection from cell and organ experiments.Methods: PIEC was exposed in human blood plasma and incubated with stachyose or PBS. Then we employed the method of MTT to determine the survival rate of cells, to investigate the protection of stachyose on PIEC. Construct a model of hyperacute rejection of in vitro perfusion pig heart. To investigate the inhibition of stachyose on hyperacute rejection. We determine the histopathobiology and immunohistology of pig hearts which perfused with or without stachyose.Results:1. The survival rate of PIEC increased as the concentration of stachyose increased.2. We have established the model of hyperacute rejection of in vitro perfusion pig heart successfully.3. The survival time of human blood plus stachyose group were longer than control group. The mean time of perfusion hearts in human blood group was （9.5±2.5） min while in human blood plus stachyose perfusion group was （46.8±8.1） min.4. Histopathological studies showed that haemorrhagia, edema, necrosis of endothelial cell, distension of blood vessel were found in human blood perfusion group while there were no obvious pathological changes in human blood plus stachyose group.5. Immunohistochemistry studies also indicated there were obvious deposit of IgG and IgM in human blood perfusion group while in human blood plus stachyose perfusion group no deposit of IgG or IgM were found.Conclusion: Stachyose can competitively bind to natural anti-Gal antibody in human blood, inhibit hyperacute rejection. Stachyose can protect PIEC and pig heart from hyperacute rejection.ObjectiveAllotransplantation, as the most effective measure to end-stage organ failure, has resulted in shortage of organs. Attention has been drawn towards xenotransplantation. Xenotransplantation from pig to human is a potential solution to the worldwide shortage of organs. However, it has many immunology barriers. The imcompatibility of blood type between human and pig may be the reason to failure of organ xenotransplantation, in addition to hyperacute rejection, acute vascular rejection and chronic rejection. The objective of this article was to determine the ABO blood antigen on pig erythrocyte.Methods1. Determine A or B antigen on pig erythrocyteEmploy the method of cell agglutination. Pig erythrocyte and anti-A or anti-B was added to the agglutination plates. The agglutination of pig erythrocyte was visible when erythrocyte distributed on the bottom of the wells to form blood dumps. While erythrocyte precipitated as a small dot as no agglutination happened, which proved that no corresponding antigen was on erythrocyte.2. Determine anti-A or anti-B in plasma of pigEmploy the method of reverse typing to determine the blood antibody in plasma of pig. The plasma of pig was pre-absorbed with packed human type O erythrocyte to remove non-specific binding from anti-human heteroagglutinins.Results1. Detection of the A or B antigen on the surface of porcine erythrocytesWe did not found the blood antigen on pig erythrocyte （n=72）.2. Determine anti-A or anti-B in plasma of pigWe determined anti-A or anti-B antibodies in plasma of 72 pigs. 37 pigs were found both anti-A and anti-B antibodies, 14 pigs were found anti-A antibodies, only 3 pigs were found anti-B antibodies, and 18 pigs were found neither anti-A nor anti-B antibodies.ConlusionFrom our experiments, we have demonstrated:Although pig erythrocytes do not express ABO blood antigen which is similar with human, blood group antibodies are in the plasma of pig. We have determined plasma of 72 pigs, 51.4% was found anti-A and anti-B antibody in their plasma; 19.4 was found anti-A antibody; 4.2% was found anti-B antibody; 25% was found neither anti-A nor anti-B antibody.更多还原