【作者】 王东梅；

【导师】 张志欣； 宋长兴；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 移植免疫， 2009， 博士

【摘要】 器官移植是治疗晚期器官衰竭患者的有效措施,但由于可用的人体器官来源有限,一直限制着其广泛应用,随着器官衰竭患者的增加,供需矛盾越来越突出。利用动物器官不失为一条很好的解决途径。异种器官移植的理想器官供体是猪。虽然,存在种属间的不相容性,但由于猪的器官在解剖与生理方面与人的器官更为相近,被认为是最适宜的异种器官来源。由于种属间关系较远,人对猪的器官会发生排斥反应,包括超急性排斥反应(hyperacute rejection,HAR),急性血管性排斥反应(acute vascular rejection,AVR),及慢性排斥反应(chronic rejection,CR)。HAR的发生主要是由于受者体内存在的天然抗体,识别猪血管内皮细胞表面的Galα1,3Gal引起的。除了人、猿和旧大陆猴,几乎所有哺乳动物细胞表面均表达Galα1,3Gal抗原。AVR的发生与抗体也有密切的关系,包括抗Gal抗体和抗非Gal抗体。对CR的研究较少,但明确的是人体内的T细胞通过直接识别和间接识别途径,被激活后引发的炎症反应在CR中起到重要的作用。克服异种移植免疫排斥的研究有很多,其中去除猪细胞表面的Galα1,3Gal抗原、通过转基因技术在猪细胞上表达人的补体成分(如CD55,CD59等)和HLA-E,G分子等、使用药物抑制补体活性和免疫排斥反应等方法都取得了明显的疗效,获得了学术界的肯定。台湾的Jang-Ming Lee等将HLA-DP\DQ基因导入猪,减少了慢性排斥阶段的细胞反应。人的MHC分子是HLA,即人白细胞抗原,分为Ⅰ,Ⅱ和Ⅲ类分子,其中Ⅰ类包括HLA-A,-B,-C,-E,-G,-F等,Ⅱ类包括HLA-DR,-DP,-DQ等,Ⅲ类位于I类区域与I类区域之间,主要有补体C2、C4A、C4B(complement component C2、C4A、C4B)等,是与HLA无关的非HLA分子,仅习惯上列为Ⅲ类分子。HLA分子在调解免疫上发挥着重要作用,其中HLA-DRB1分子的作用更为突出。本研究的目的是探索HLA-DRB1分子在猪细胞上表达的可行性,为以后研究利用HLA-DRB1分子抑制免疫排斥反应打下基础。第一部分:HLA-DRB1*09的筛选目的:从人群中筛选到HLA-DRB1位点是09的个体。方法:使用HLA基因分型技术PCR-SSO和PCR-SBT,对人群的HLA-DRB1位点进行分型。结果:确定了表达HLA-DRB1*09的个体。结论:HLA分子生物学分型技术有效、快捷的找到了HLA-DRB1*09的个体,其HLA-DRB1基因型是090102和0101。第二部分:HLA-DRB1*0901及HLA-DRA基因真核细胞双表达载体的构建及鉴定目的:构建HLA-DRB1*0901及HLA-DRA基因真核细胞双表达载体,并对其是否表达鉴定。方法:用RT-PCR的方法从人外周血淋巴细胞的mRNA中逆转录扩增出HLA-DRB1*0901和HLA-DRA的cDNA, HLA-DRA经BglⅡ酶切插入真核表达载体pVITRO2的MCS1中,同时确认正反向,再在pVITRO2的MCS2中连入BamHⅠ酶切的HLA-DRB,确认正反向。将双表达载体转染猪肾细胞,western-blotting鉴定是否表达。结果:酶切和PCR分析鉴定,得到了pVITRO2-DRA-DRB1*0901双表达载体,western-blotting鉴定出有表达。结论:双表达载体pVITRO2可以在猪肾细胞中表达HLA-DRB1*0901。更多还原

【Abstract】 The increasing success in allotransplantation has generated a severe shortage of organs available for transplantation. The alternative to using animals as organ donors has focused on the pig as a source for xenegrafts. Although interspecies incompatibilities exist, pig organs are considered to be most suited for xenotransplantation because of similarities in size and physiology of pigs and humans.Because of interspecies incompatibilities, there will happen pig-to-human xenograft rejection, including hyperacute rejection, acute vascular rejection, and chronic rejection. Hyperacute rejection is mediated by nature antibody existed in recipient, which react with the galactosylα1,3-galactose (αGal) epitopes on the endothelium of the graft. With the exception of humans, apes, and Old World monkeys, almost all mammalian cells express galactosylα1,3-galactose antigen. Acute vascular rejection is medicated by anti-Gal and anti-non Gal antibodies. There is little knowledge about chronic rejection, but it has been demonstrated that the porcine MHC molecules can effectively induce a strong human T-cell response, through direct or indirect antigen recognition. Many researches were done on overcoming xenotransplantation immuno-rejection, such as, knocking outαGal antigen of procine cells, genetic modifying the pig organs for human complement regulator, and using immunosuppress drugs. Researchers of Taiwan University Hospital have produced the human HLA DPw0401 transgenic pig and found HLA DPw0401 molecule can attenuate human-to-pig xenogenic cellular response. The genetic loci involved in the rejection of foreign organs are known as the major histocompatibility complex (MHC).The human MHC is called human leukocyte antigen(HLA) which is divided into HLA classⅠ、classⅡand classⅢ. The HLA classⅠmolecules include HLA-A、-B、-C、-E、-F、-G,etc. The HLA classⅡmolecules include DR、DP、DQ. The HLA classⅢmolecules include C2、C4A、C4B which is irrelevant with HLA. The HLA molecules play an important role at regulating immunoreaction.The aim of this study is to investigate the expression of human HLA-DRB1 molecule on pig kidney cell. This research will lay foundation for further investigation the role of HLA-DRB1 on xenograft immunological rejection.First part: identified HLA-DRB1*09Objective: To identify sample whose HLA-DRB1 genotype is 09.Methods: The samples’genomic DNA was extracted by salting-out method and typed by PCR-SSO and PCR-SBT.Result: Individual whose HLA-DRB1 genotype is 09 was found.Conclusion: HLA typing methods can quick and accurately identify HLA genotype. The individual’s HLA-DRB1 locus is 090102/010101.Second part: Construction and identification of the eukaryotic co-expression vector of human HLA-DRB1*0901 and HLA-DRA with pVITRO2 Objective: To construct the eukaryotic co-expression vector of human HLA-DRB1*0901 and HLA-DRA with Pvitro2 and identify its expression in kidney cell of porcine(LLC-PK1).Methods: RT-PCR was used to amplify the HLA-DRB1*0901 and HLA-DRA cDNA from total RNA of leukocyte in peripheral blood. The HLA-DRB1*0901 DNA fragment(digested with BglⅡ) was inserted into the multiple cloning site 1 of vector pVITRO2. after identified its direction, the HLA-DRB1*0901 DNA fragment(digested with BamHⅠ) was inserted into the multiple cloning site2 of vector pVITRO2-dra. Then, the co-expression vector pVITRO2-dra-drb was transfected into the LLC-PK1, and western-blotting was used to identified its expression.Results: The restriction endonucleases digestion and PCR suggested the co- expression vector pVITRO2-dra-drb was constructed successfully. Western-blotting identified the protein HLA-DRB1. Conclusion: The co-expression vector pVITRO2-dra-drb can express human HLA-DRB1*0901 in LLC-PK1.更多还原