人HLA基因DRB1*0901在猪肾细胞中的表达及鉴定
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。
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*09
Objective: 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.
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。
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*09
Objective: 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.
引文
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11 Salerno CT, Kulick DM, Yeh CG, et al. A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation. Xenotransplantation, 2002,9(2):125-134.
12 Poirier N, Blancho G. Recombinant human C1-inhibitor inhibits cytotoxicity induced by allo- and xenoantibodies. Transplant Proc. 2008, 40(2):581-583.
13 Banz Y, Rieben R. Endothelial cell protection in xenotransplantation: looking after a key player in rejection. Xenotransplantation, 2006,13(1):19-30.
14 Carrington CA, Dos Santos, Cruz G. Effect of cell surface concentration of human DAF on transgenic pig aortic endothelial cells on the degree of protection afforded against human complement deposition. Xenotransplantation, 2001, 8(2): 100-105.
15 Shiraishi M, Oshiro T, Nozato E, et al. Adenovirus-mediated gene transfer of triple human complement regulating proteins(DAF, MCP and CD59) in the xenogeneic porcine-to-human transplantation modelPart II: xenogeneic perfusion of the porcine liver in vivo. Transpl Int, 2002, 15(5): 212-219.
16 Fisicaro N, Aminian A, Hinchliffe SJ, et al. The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement. Transplantation, 2000,70(6):963-968.
17 Morgan BP, Berg CW, Harris CL. ''Homologous restriction'' in complement lysis: roles of membrane complement regulators. Xenotransplantation, 2005, 12(4):258-265.
18 Ezzelarab M, Hara H, Busch J, et al. Antibodies directed to pig non-Gal antigens in naive and sensitized baboons. Xenotransplantation, 2006, 13(5):400-407.
19 Galili U. The nature of elicited xenoantibodies. Transplant Proc, 2001, 33(1-2): 688-689.
20 Matsunami K, Miyagawa S, Nakagawa K, et al Molecular cloning of pigGnT-I and I.2: an application to xenotransplantation. Biochem Biophys Res Commun, 2006, 343(3):677-683.
21 Robson SC, Cooper DK, d'Apice AJ. Disordered regulation of coagulation and platelet activation in xenotransplantation. Xenotransplantation, 2000,7(3):166-176.
22 d'Apice AJ. Is xenotransplantation of vascularized organs just too hard? Xenotransplantation, 2006, 13(3):182-185.
23 Lee KF, Salvaris EJ, Roussel JC,et al. Recombinant pig TFPI efficiently regulates human tissue factor pathways. Xenotransplantation, 2008,15(3):191-197.
24 Kim YT, Lee HJ, Lee SW, et al. Pre-treatment of porcine pulmonary xenograft with desmopressin: a novel strategy to attenuate platelet activation and systemic intravascular coagulation in an ex-vivo model of swine-to-human pulmonary. Xenotransplantation, 2008, 15:27-35.
25陈实.移植免疫学.武汉:湖北科学技术出版社,1998:323-325.
26 Kharbanda RK, Deanfield JE. Functions of the healthy endothelium. Coron Artery Dis, 2001, 12: 485-491.
27 Smolenski RT, Khalpey Z, Osborne FN,et al. Species differences of endothelial extracellular nucleotide metabolism and its implications for xenotransplantation. Pharmacol Rep, 2006, 58 Suppl:118-125.
28 Lilienfeld BG, Crew MD, Forte P,et al. Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity. Xenotransplantation, 2007, 14:126-134.
29 Cooper DK. Is xenotransplantation a realistic clinical option? Transplant Proc, 1992, 24: 2393 - 2396.
30 Dalmasso AP, Benson BA, Johnson JS, et al. Resistance against the membrane attack complex of complement induced in porcine endothelial cells with a Gal alpha(1-3)Gal binding lectin: up-regulation of CD59 expression. J Immunol, 2000, 164(7):3764-73.
31 Suhr BD, Black SM, Guzman-Paz M, et al. Inhibition of the membrane attack complex of complement for induction of accommodation in the hamster-to-rat heart transplant model. Xenotransplantation,2007,14(6):572-579.
32 Dawson JR,Vidal AC, Malyguine AM, Natural killer cell-endothelial cell interactions in xenotransplantation. Immunol Res, 2000,22:165-172.
33 ChristiansenD, Monhhtouris E, Milland J, Recognition of a carbohydrate xenoepitope by human NKRPIA(CD161) .Xenotransplantation,2006,13(5):440-446.
34 Crew MD, Play it in E or G: utilization of HLA-E and -G in xenotransplantation. Xenotransplantation, 2007, 14(3):198-207.
35 Costa C, Barber DF, Fodor WL. Human NK cell-mediated cytotoxicity triggered by CD86 and Gal alpha 1,3-Gal is inhibited in genetically modified porcine cells. J Immunol, 2002 , 168(8):3808-3816.
36 Lilienfeld BG, Garcia-Borges C, Crew MD,et al. Porcine UL16-binding protein 1 expressed on the surface of endothelial cells triggers human NK cytotoxicity through NKG2D. J Immunol. 2006,177(4):2146-2152.
37沈佰华,谢晋,李宁丽,等.人特异性T细胞系间接识别猪抗原.上海免疫学杂志, 2001,21(4):210-212.
38 Buhler L, Cooper DK, Models of xenotransplantation tolerance. Curr.Opin.Organ Transplantation, 2003,8:83-88.
39 Ohdan H,Yang YG, Swenson KG, et al. T cell and B cell tolerance to GALaL PHA 1,3 GAL–expressing heart xenografts is achieved in alpha1, 3- galactosyltransferase-deficient mice by nonmyeloablative induction of mixed chimerism . Transplantation, 2001, 71:1532-1542.
40 Mulley WR, Li YQ, Wee JL, et al. Local expression of IDO,either alone or in combination with CD40Ig,IL10 or CTLAIg inhibits indirect xenorejection responses. Xenotransplantation,2008,15:174-183.
41 Séveno C, Coulon F, Haspot F,et al. Induction of regulatory cells and control of cellular but not vascular rejection by costimulation blockade in hamster-to-rat heart xenotransplantation. Xenotransplantation. 2007,14(1):25-33.
42 Jang-Ming Lee, Ching-Fu Tu, Pei-Wen Yang, et al. Reduction of human-to-pig cellular response by alteration of porcine MHC with human HLA DPW0401 exogenes. Transplantation ,2002, 73(2):193-197.
43 Wang J, Jiang S, Shi H, et al. Prolongation of corneal xenotransplant survival by T-cell vaccination- induced T-regulatory cells. Xenotransplantation, 2008 , 15(3):164-173.
44 Gianello P, Dufrane D. Correction of a diabetes mellitus type 1 on primate with encapsulated islet of pig pancreatic transplant. Bull Mem Acad R Med Belg,2007, 162(10-12):439-49;
45 Zhu M, Zhang W, Liu F,et al. Resistance to anti-xenogeneic response by combining alpha-Gal silencing with HO-1 upregulation. Transpl Immunol, 2008,19(3-4):202-208.
46 Shimizu A, Hisashi Y, Kuwaki K, et al. Thrombotic microangiopathy associated with humoral rejection of cardiac xenografts from alpha1,3-galactosyltransferase gene-knockout pigs in baboons. Am J Pathol. 2008 ,172(6):1471-1481.
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1 Cascalho M, Platt JL. The immunological barrier to xenotransplantation. Immunity, 2001, 14(4):437-446.
2 Julie M.Illand, Dale Christiansen ,MauRO S. Sandrin,α1,3-Galactosyltransferase knockout pigs are available for xenotransplantation:Are glycosyltransferases still relavant? Immunology and cell Bio,2005, 83.687-693
3 Larsen RD, Rivera-Marrero CA, Ernst LK, et al. Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3) -galactosyltransferase cDNA. J Biol Chem, 1990, 265(12):7055-7061.
4 Luo Y, Wen J, Luo C, et al. Pig xenogeneic antigen modification with green coffee bean alpha-galactosidase. Xenotransplantation, 1999, 6(4):238-248.
5 Sandrin MS, Fodor WL, Mouhtouris E,et al. Enzymatic remodelling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement- mediated cytolysis. Nat Med,1995, 1(12):1261-1267.
6 Lai L, Kolber-Simonds D, Park KW, et al. Production of alpha- 1,3- galactosyltransferase knockout pigs by nuclear transfer cloning. Science, 2002, 295(5557): 1089-1092.
7 Zhu M, Wang SS, Xia ZX, Cao RH, et al. Inhibition of xenogeneic response in porcine endothelium using RNA interference. Transplantation, 2005, 15;79(3):289-296.
8 Watt.SR, Jeff M.Betlhauser, Augenstein ML et al. Direct and Rapid Modification of a porcine Xenoantigen Gene. Transplantation, 2006, 82(7).975-978.
9 Eisen A,Camerini-Otero R D, A recombinase from Drosophila melanogaster embryos PNAS ,1988, 85:7481-7485
10 Brandl U, Erhardt M, Michel S ,et al. Soluble Galα(1,3)Gal conjugate combined with hDAF preserves morphology and improves function of cardiac xenografts. Xenotransplantation,2007, 14:323-332.
11 Salerno CT, Kulick DM, Yeh CG, et al. A soluble chimeric inhibitor of C3 and C5 convertases, complement activation blocker-2, prolongs graft survival in pig-to-rhesus monkey heart transplantation. Xenotransplantation, 2002,9(2):125-134.
12 Poirier N, Blancho G. Recombinant human C1-inhibitor inhibits cytotoxicity induced by allo- and xenoantibodies. Transplant Proc. 2008, 40(2):581-583.
13 Banz Y, Rieben R. Endothelial cell protection in xenotransplantation: looking after a key player in rejection. Xenotransplantation, 2006,13(1):19-30.
14 Carrington CA, Dos Santos, Cruz G. Effect of cell surface concentration of human DAF on transgenic pig aortic endothelial cells on the degree of protection afforded against human complement deposition. Xenotransplantation, 2001, 8(2): 100-105.
15 Shiraishi M, Oshiro T, Nozato E, et al. Adenovirus-mediated gene transfer of triple human complement regulating proteins(DAF, MCP and CD59) in the xenogeneic porcine-to-human transplantation modelPart II: xenogeneic perfusion of the porcine liver in vivo. Transpl Int, 2002, 15(5): 212-219.
16 Fisicaro N, Aminian A, Hinchliffe SJ, et al. The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement. Transplantation, 2000,70(6):963-968.
17 Morgan BP, Berg CW, Harris CL. ''Homologous restriction'' in complement lysis: roles of membrane complement regulators. Xenotransplantation, 2005, 12(4):258-265.
18 Ezzelarab M, Hara H, Busch J, et al. Antibodies directed to pig non-Gal antigens in naive and sensitized baboons. Xenotransplantation, 2006, 13(5):400-407.
19 Galili U. The nature of elicited xenoantibodies. Transplant Proc, 2001, 33(1-2): 688-689.
20 Matsunami K, Miyagawa S, Nakagawa K, et al Molecular cloning of pigGnT-I and I.2: an application to xenotransplantation. Biochem Biophys Res Commun, 2006, 343(3):677-683.
21 Robson SC, Cooper DK, d'Apice AJ. Disordered regulation of coagulation and platelet activation in xenotransplantation. Xenotransplantation, 2000,7(3):166-176.
22 d'Apice AJ. Is xenotransplantation of vascularized organs just too hard? Xenotransplantation, 2006, 13(3):182-185.
23 Lee KF, Salvaris EJ, Roussel JC,et al. Recombinant pig TFPI efficiently regulates human tissue factor pathways. Xenotransplantation, 2008,15(3):191-197.
24 Kim YT, Lee HJ, Lee SW, et al. Pre-treatment of porcine pulmonary xenograft with desmopressin: a novel strategy to attenuate platelet activation and systemic intravascular coagulation in an ex-vivo model of swine-to-human pulmonary. Xenotransplantation, 2008, 15:27-35.
25陈实.移植免疫学.武汉:湖北科学技术出版社,1998:323-325.
26 Kharbanda RK, Deanfield JE. Functions of the healthy endothelium. Coron Artery Dis, 2001, 12: 485-491.
27 Smolenski RT, Khalpey Z, Osborne FN,et al. Species differences of endothelial extracellular nucleotide metabolism and its implications for xenotransplantation. Pharmacol Rep, 2006, 58 Suppl:118-125.
28 Lilienfeld BG, Crew MD, Forte P,et al. Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity. Xenotransplantation, 2007, 14:126-134.
29 Cooper DK. Is xenotransplantation a realistic clinical option? Transplant Proc, 1992, 24: 2393 - 2396.
30 Dalmasso AP, Benson BA, Johnson JS, et al. Resistance against the membrane attack complex of complement induced in porcine endothelial cells with a Gal alpha(1-3)Gal binding lectin: up-regulation of CD59 expression. J Immunol, 2000, 164(7):3764-73.
31 Suhr BD, Black SM, Guzman-Paz M, et al. Inhibition of the membrane attack complex of complement for induction of accommodation in the hamster-to-rat heart transplant model. Xenotransplantation,2007,14(6):572-579.
32 Dawson JR,Vidal AC, Malyguine AM, Natural killer cell-endothelial cell interactions in xenotransplantation. Immunol Res, 2000,22:165-172.
33 ChristiansenD, Monhhtouris E, Milland J, Recognition of a carbohydrate xenoepitope by human NKRPIA(CD161) .Xenotransplantation,2006,13(5):440-446.
34 Crew MD, Play it in E or G: utilization of HLA-E and -G in xenotransplantation. Xenotransplantation, 2007, 14(3):198-207.
35 Costa C, Barber DF, Fodor WL. Human NK cell-mediated cytotoxicity triggered by CD86 and Gal alpha 1,3-Gal is inhibited in genetically modified porcine cells. J Immunol, 2002 , 168(8):3808-3816.
36 Lilienfeld BG, Garcia-Borges C, Crew MD,et al. Porcine UL16-binding protein 1 expressed on the surface of endothelial cells triggers human NK cytotoxicity through NKG2D. J Immunol. 2006,177(4):2146-2152.
37沈佰华,谢晋,李宁丽,等.人特异性T细胞系间接识别猪抗原.上海免疫学杂志, 2001,21(4):210-212.
38 Buhler L, Cooper DK, Models of xenotransplantation tolerance. Curr.Opin.Organ Transplantation, 2003,8:83-88.
39 Ohdan H,Yang YG, Swenson KG, et al. T cell and B cell tolerance to GALaL PHA 1,3 GAL–expressing heart xenografts is achieved in alpha1, 3- galactosyltransferase-deficient mice by nonmyeloablative induction of mixed chimerism . Transplantation, 2001, 71:1532-1542.
40 Mulley WR, Li YQ, Wee JL, et al. Local expression of IDO,either alone or in combination with CD40Ig,IL10 or CTLAIg inhibits indirect xenorejection responses. Xenotransplantation,2008,15:174-183.
41 Séveno C, Coulon F, Haspot F,et al. Induction of regulatory cells and control of cellular but not vascular rejection by costimulation blockade in hamster-to-rat heart xenotransplantation. Xenotransplantation. 2007,14(1):25-33.
42 Jang-Ming Lee, Ching-Fu Tu, Pei-Wen Yang, et al. Reduction of human-to-pig cellular response by alteration of porcine MHC with human HLA DPW0401 exogenes. Transplantation ,2002, 73(2):193-197.
43 Wang J, Jiang S, Shi H, et al. Prolongation of corneal xenotransplant survival by T-cell vaccination- induced T-regulatory cells. Xenotransplantation, 2008 , 15(3):164-173.
44 Gianello P, Dufrane D. Correction of a diabetes mellitus type 1 on primate with encapsulated islet of pig pancreatic transplant. Bull Mem Acad R Med Belg,2007, 162(10-12):439-49;
45 Zhu M, Zhang W, Liu F,et al. Resistance to anti-xenogeneic response by combining alpha-Gal silencing with HO-1 upregulation. Transpl Immunol, 2008,19(3-4):202-208.
46 Shimizu A, Hisashi Y, Kuwaki K, et al. Thrombotic microangiopathy associated with humoral rejection of cardiac xenografts from alpha1,3-galactosyltransferase gene-knockout pigs in baboons. Am J Pathol. 2008 ,172(6):1471-1481.