摘要
同种异体移植已在临床广泛开展，并成为根治许多终末期器官功能衰竭疾病的唯一手段。目前面临的问题是：人器官移植物来源十分有限，且获得供体的时机极为随机，以至临床需求缺口日益扩大，并成为制约临床开展移植术的主要因素。为解决供体器官严重短缺的难题，应用异种动物(主要是猪)为供体的异种移植成为近年移植免疫学领域的研究热点之一。
由于灵长类动物(包括人)体内存在针对猪组织细胞表面α-半乳糖苷的天然抗体，故猪→人异种移植中，人天然抗体可与猪组织细胞表面抗原结合，通过激活补体而导致供者移植物细胞溶解，并引发超急性排斥反应。国内外均已报道：通过抑制受者补体系统激活等手段，可成功克服超急性异种移植排斥反应。因此，异种移植后的急性血管性排斥反应和急性细胞性排斥反应成为人们关注的重点。目前有关异种移植的实验研究主要集中于如下领域：①猪的主要组织相容性复合体(SLA)；②NK细胞ADCC效应对异种靶细胞的杀伤作用，以及人CD4~+和CD8~+T细胞介导异种细胞移植排斥反应的机制。
本课题分析中国猪种SLA基因结构特征，以及人NK细胞杀伤异种靶细胞过程中的胞吐作用及其机制，其立题依据是：
1．猪MHC(SLA)分子是引发急性和慢性异种移植排斥反应的关键抗原，分析比较SLA与人HLA复合体遗传结构的异同，有助于探讨异种移植排斥反应的发生机制，并为选择猪种和制定临床干预方案提供重要线索。
2．人NK细胞是参与异种移植排斥反应的重要效应细胞，其被靶细胞(即猪血管内皮细胞)触发，通过杀伤性胞吐(脱颗粒)过程而释放颗粒酶、穿孔素等，可对猪血管内皮细胞发挥杀伤效应，此乃猪→人异种器官移植急性排斥反应中最早发生和最关键的病理过程。
一、异种移植备选中国猪种的经典SLA基因序列分析
近二十余年，国外已分析了多个实验猪种的SLA复合体及其抗原系统，为探讨人类免疫系统识别SLA抗原的机制奠定了理论基础。
我国是猪种资源最为丰富的国家，猪种遗传性状稳定，为筛选合适的供体猪种提供了有利条件。为探讨我国猪种SLA基因结构及其在猪→人异种器官移植中的作用，本
课题选择中国不同地区3个常见实验用猪种—湖北中华大白猪封闭群、海南五指山猪
近交系和云南版纳小耳猪近交系为研究对象，根据Genbank中NIH小型猪SLAI类和
n类基因序列设计引物，采用RT一PCR方法获得相应基因的cDNA产物，电泳鉴定后纯
化，对PCR产物进行直接测序或克隆入测序T载体后进行双向测序，获得中国猪种SLA
I类和n类基因cDNA序列，进而借助DNA Tools 5.0软件包和GCG Ver. 2.9软件包进
行相关比较和分析。本课题的实验结果为:
1 .3个中国猪种SLA一DRA基因为高度保守序列，无多态性，与NIH小型猪在核
昔酸和氨基酸水平的同源性均为100%;3个中国猪种其他的经典SLAI类和n类基因
(PI、P14、DQA、DQB、D邓)均具多态性，与NIH小型猪在核昔酸水平同源性高
于90%，在氨基酸水平同源性高于87%。结果提示:受试中国猪种Pl、P14、DQA、
DQB、DRB基因分别为相应基因座位上的新等位基因。
2.中国人群中HLAI类高频等位基因HLA一A*0 201与中国猪种P1和P14基因产
物的氨基酸序列同源性仅为63%，但在MHC分子多态样al和a2功能区中与抗原肤锚
着位相对应的位点，其氨基酸残基基本相同。结果提示:不同种属动物MHC分子肤结
合区的差异较小，具有一定保守性。
3 .SLA一Pl、SLA一P14和HLA一A*0201产物中参与同人CDS结合所必需的关键氨
基酸残基，仅有两个存在差异，即:第223位丙氨酸(Ala)变为缴氨酸(Val);第
235位丙氨酸(Ala)变为赖氨酸(Lys)。SLA一Pl、SLA一P14和HLA一A*0201产物中参
与同人CDS结合所必需的关键氨基酸残基的高度相似性，提示SLAI类分子可跨越种
属界限而与人CDS分子结合，从而发挥相应生物学作用。
4.两个近交系猪种SLA一DRA和人HLA一DRA产物与CD4分子结合所必需的关键
氨基酸残基基本相同，仅在两个位点发生相同变异，即:第125位精氨酸(Arg)变为
甘氨酸(Gly);第129位撷氨酸(Val)变为异亮氨酸(I le)。而SLA一DRB分子参与
结合CD4所必需的关键氨基酸残基与人类完全相同，此现象在国内外其他猪种SLA序
列研究中尚未见报道。上述结果，即SLAn类分子和人HLAn类分子结构具有高度相
似性，进一步提示:中国猪种SLAH类分子可跨越种属界限而与人CD4分子结合，并
发挥相应生物学作用。
5.中国猪种SLAI类分子(P1和P14)缺乏与NK细胞表面杀伤细胞抑制性受体
(KIR)结合的关键氨基酸残基。提示:由于缺乏KIR的配基序列，中国猪种组织细胞
难以启动NK细胞的负调节信号，故猪源移植物易遭人(受者)NK细胞杀伤。由此，
本课题从新的角度阐明了猪~人异种移植中猪源组织细胞受损的可能机制。
综上所述，通过在分子遗传学水平分析、比较SLA和HLA基因结构，发现海南五
指山猪近交系和云南版纳小耳猪近交系SLA与人HLA遗传背景具有高度相似性，但两
个中国猪种相关基因序列发生变异的氨基酸残基与NIH小型猪有所不同。本课题获得
了中国主要实验猪种SLA基因结构的初步信息，通过进一步阐明所发生变异的确切生
物学作用，可望为中国猪种用于人类
The success of organ transplantation has led to an ever-increasing shortfall between the demand for organs and the supply in late 40 years. This has given more chances to extensive investigation of the possible use of animals, especially the pigs, as organ donors.
Hyperacute xeno-rejection was overcome through inhibiting the activation of the complement system. The acute vascular rejection and acute cellular rejection become more and more important in xenotransplantation rejection research. The new focus were Swine Leukocyte Antigen (SLA) and the mechanisms of antibody dependent cell mediated cytotoxicity (ADCC) induced by natural killer cells (NK) as well as the rejection mechanism induced by CD4+ and CD8+ T cells. The NK cells cytotoxicity to porcine endothelial cell (PEC) is the first stage and also the most important point in the delayed xeno-rejection. NK cells possess granules containing perform and granzyme that are released upon activation to mediate cytotoxicity in target PECs
Sequence Analysis of Classical Swine Leukocyte Antigens (SLA) Class I and Class II Molecules in Chinese Pigs
Foreign research groups analyzed some SLA antigen systems of different pig strains since 1980s companied with progress of molecular technology. They have provided some useful evidences to explain the MHC restriction in recognition process by human immune cells. China is rich in swine resources and has more than 100 natural strains and most of them have stale characteristics.
To screen a proper pig strain for xenograft, Hubei White Pigs (HBWP), inbred Wuzhishan miniature pigs (WZSP) and inbred Yunnan Bana miniature pigs (BMI) were taken as the subjects. The classical SLA class I molecules (P1 and P14) and class II molecules (DQA DQB DRA DRB) were investigated by reverse transcription polymerase chain reaction (RT-PCR) to obtain cDNA products. The purified products were sequenced and compared with the data of NIH miniature pigs as well as those relevant to high frequency genes of HLA in Genbank for homology analysis and some other related comparisons.
The findings indicated that three Chinese pigs are highly homologous (>90%) with the inbred NIH miniature swine in characterization of porcine MHC classical class I and class II molecules. While, the obtained DRA sequence is completely same and is confirmed that DRA molecule is highly conserved in the porcine genes. So, the other sequences obtained are the new alleles in relevant loci (P1, P14, DQA, DQB and DRB). The homologous comparison with the HLA-A*0201 was about 63% in amino acid level. The greatest degree of polymorphism is within the a1 and a2 domains, while the others are highly conserved. The various series SLA may have less difference in this binding domain. The amino acids in a2 and a3 domains those were responsible for binding with human CD8 to MHC class I were largely conserved, only two critical residues were altered, which were a Ala-Val change at position 223 and a Ala-Lys change at position 235. And, correlative comparison of the amino acids for binding of human CD4 to MHC class II revealed that only two amino acids responsible for binding of CD4 were altered, a Arg-Gly change at position 125 and the other change Val-Ile at position 129. Therefore, the key residues of new DRB for binding of CD4 are the same as those in HLA-DRB*09012 absolutely. There was no similar report in related research before. The high similarity of SLA class I and II molecules show that they would bind with human CD8 or CD4 molecules and play some similar roles. None of the binding sequences responsible for killer inhibitory receptors were present in the porcine molecules in this study. It would result great killing of pig cells by human NK cells in xeno-rejection.
In a word, SLA molecules of inbred WZSP and BMI are highly homologous with HLA relevant molecules. And, they have some specific and stable mutation characteristic. However, there are no certain answer to say these two inbred strains would be the best animal model in future porcine-human xenotransplantation just according to l
引文
[1] Cascalho M, Platt JL. Xenotransplantation and other means of organ replacement. Nat Rev Immunol. 2001, 1(2): 154-160.
[2] Sim KH, Marinov A, Levy GA. Xenotransplantation: A potential solution to the critical organ donor shortage. Can J Gastroenterol. 1999, 13(4): 311-318.
[3] Platt JL, Lin SS. The future promises ofxenotransplantation. Ann N Y Acad Sci. 1998, 30(862): 5-18.
[4] Minanov OP, Itescu S, Michler RE. Recent advances and the potential for clinical use of xenotransplantation. Curr Opin Cardiol. 1996, 11(2): 214-220.
[5] Bach FH, Winkler H, Wdghton CJ, Robson SC, Stuhlmeier K, Ferran C. Xenotransplantation: a possible solution to the shortage of donor organs. Transplant Proc. 1996, 28(1): 416-417.
[6] Fryer JP, Leventhal JR, Matas AJ. The emergence of xenotransplantation. Transpl Immunol. 1995, 3(1): 21-31.
[7] Dunning JJ, White D J, Wallwork J. The rationale for xenotransplantation as a solution to the donor organ shortage. Pathol Biol (Paris). 1994, 42(3): 231-235.
[8] Somerville CA, d'Apice AJ. Future directions in transplantation: xenotransplantation. Kidney Int Suppl. 1993, 42: S112-121.
[9] Knosalla C, Gollackner B, Dor FJ, Cooper DK. Therapeutic interventions in xenotransplantation. Curr Drug Targets Cardiovasc Haematol Disord. 2002, 2(2): 105-119.
[10] Cooper DK, Gollaekner B, Knosalla C, Teranishi K. Xenotransplantation—how far have we come? Transpl Immunol. 2002, 9(2-4): 251-256.
[11] Lin SS. Therapeutic strategies for xenograft rejection. J Card Surg. 2001, 16(5): 374-382.
[12] Perico N, Benigni A, Remuzzi G. Xenotransplantation in the 21st century. Blood Purif. 2002, 20(1): 45-54.
[13] Igaz P. Recent strategies to overcome the hyperacute rejection in pig to human
xenotransplantation. Yale J Biol Med. 2001, 74(5): 329-340.
[14] Alwayn IP, Basker M, Buhler L, Cooper DK. The problem of anti-pig antibodies in pig-to-primate xenografting: current and novel methods of depletion and/or suppression of production of anti-pig antibodies. Xenotransplantation. 1999, 6(3): 157-168.
[15] Levy GA, Marsden P, Zhong R, Cole EH, Grant D. Strategies to prevent thrombosis in xenotransplants. Transplant Proc. 1998, 30(5): 2458-2460.
[16] van den Bogaerde J, White DJ. Xenogeneic transplantation. Br Med Bull. 1997, 53(4): 904-920.
[17] Hancock WW. Beyond hyperacute rejection: strategies for development of pig--＞primate xenotransplantation. Kidney Int Suppl. 1997, 58: S36-40.
[18] Logan JS. Prospects for xenotransplantation. Curr Opin Immunol. 2000, 12(5): 563-568.
[19] Cozzi E, Masroor S, Soin B, Vial C, White DJ. Progress in xenotransplantation. Clin Nephrol. 2000, 53(4): suppl 13-18.
[20] Lanza RP, Cooper DK. Xenotransplantation of cells and tissues: application to a range of diseases, from diabetes to Alzheimer's. Mol Med Today. 1998, 4(1): 39-45.
[21] Platt JL. Xenotransplantation: recent progress and current perspectives. Curr Opin Immunol. 1996, 8(5): 721-728.
[22] Dorling A. Clinical xenotransplantation: pigs might fly? Am J Transplant. 2002, 2(8): 695 -700.
[23] Niemann H. Current status and perspectives for the generation of transgenic pigs for xenotransplantation. Ann Transplant. 2001, 6(3): 6-9.
[24] Buhler L, Cooper DK. The pig as a source of cardiac xenografts. J Card Surg. 2001, 16(5): 345-356.
[25] Cooper DK, Gollackner B, Sachs DH. Will the pig solve the transplantation backlog? Annu Rev Med. 2002, 53: 133-147.
[26] Morris PJ. Xenotransplantation. Br Med Bull, 1999, 55(2): 446-459.
[27] Logan JS, Sharma A. Potential use of genetically modified pigs as organ donors for transplantation into humans. Clin Exp Pharmacol Physiol. 1999, 26(12): 1020-1025.
[28] Lavitrano M, Frati L. Xenotransplantation: state of the art. Forum (Genova). 1999, 9 (3 Suppl 3): 74-83.
[29] Wolf P, Meyer C, Boudjema K, Kieny R, Cinqualbre J, Jaeck D, Andre E, Herrenschmidt N, Azimzadeh A. The pig as a model in liver xenotransplantation. Vet Res. 1997, 28(3): 217-222.
[30] Swindle MM. Considerations of specific pathogen-free swine (SPF) in xenotransplantation. J Invest Surg. 1996, 9(4): 267-271.
[31] 陈慰峰。医学免疫学，2000，第三版，人民卫生出版社，第53-57页。
[32] 龚非力。医学免疫学，2000，人民卫生出版社，第56-57页。
[33] 周光炎。免疫学原理，2000，上海科学技术文献出版社，第63-68页。
[34] Vaiman M, Renard C, Lafage P, Ameteau J, Nizza P. Evidence for a histocompatibility system in swine (SL-A). Transplantation, 1970, 10 (2): 155-164.
[35] Sachs DH, Leight G, Cone J, Schwarz S, Stuart L, Rosenberg S. Transplantation in miniature swine. Ⅰ. Fixation of the major histocompatibility complex. Transplantation. 1976, 22 (6): 559-567.
[36] Rabin M, Fries R, Singer D, Ruddle FH. Assignment of the porcine major histocompatibility complex to chromosome 7 by in situ hybridization. Cytogenet Cell Genet. 1985, 39(3): 206-209.
[37] Smith TP, Rohrer GA, Alexander LJ, Troyer DL, Kirby-Dobbels KR, Janzen MA, Cornwell DL, Louis CF, Schook LB, Beattie CW. Directed integration of the physical and genetic linkage maps of swine chromosome 7 reveals that the SLA spans the centromere. Genome Res. 1995, 5(3): 259-271.
[38] Chardon P, Renard C, Vaiman M. The major histocompatibility complex in swine. Immunol Rev, 1999, 167(3): 179-192.
[39] Lunney JK and Sachs DH. Transplantation Immunology.1995, Wiley-Liss Inc. pages 339-345.
[40] Chardon P, Vaiman M, Kirszenbaum M, Geffrotin C, Renard C, Cohen D. Restriction fragment length polymorphism of the major histocompatibility complex of the pig. Immunogenetics. 1985, 21 (2): 161-171.
[41] Singer DS, Ehrlich R, goliding H, Satz L, Parent L, Rudikof S. Structure and expression of class Ⅰ MHC gene in the miniature swine. In: Molecular biology of the major hiscompatibility complex of domestic animal species. 1987, pages 53-62.
[42] Sachs DH, Germana S, el-Gamil M, Gustafsson K, Hirsch F, Pratt K. Class Ⅱ genes of miniature swine. Ⅰ. Class Ⅱ gene characterization by RFLP and by isolation from a genomic library. Immunogenetics. 1988, 28(1): 22-29.
[43] Viza D, Sugar JR, Binns RM. Lymphocyte stimulation in pigs: evidence for the existence of a single major histocompatibility locus, PL-A. Nature. 1970, 227(261): 949-950.
[44] Sullivan JA, Oettinger HF, Sachs DH, Edge AS. Analysis of polymorphism in porcine MHC class Ⅰ genes: alterations in signals recognized by human cytotoxic lymphocytes. J Immunol. 1997, 159(5): 2318-2326.
[45] Hosokawa T, Tanioka Y, Tanigawa M, Matsumoto Y, Onodera T, Matsumoto Y. Cloning and characterization of a new swine MHC (SLA) class Ⅱ DQB allele. J Vet Med Sci. 1998, 60(6): 725-729.
[46] Aida Y, Niimi M, Asahina M, Okada K, Nakai Y, Ogimoto K.Identification of a new bovine MHC class Ⅱ DRB allele by nucleotide sequencing and an analysis of phylogenetic relationships. Biochem Biophys Res Commun. 1995, 209(3): 981-988.
[47] Gustafsson K, Germana S, Hirsch F, Pratt K, LeGuern C, Sachs DH. Structure of miniature swine class Ⅱ DRB genes: conservation of hypervariable amino acid residues between distantly related mammalian species. Proc Natl Acad Sci USA. 1990, 87(24): 9798-9802.
[48] Salter RD, Benjamin RJ, Wesley PK, Buxton SE, Garrett TP, Clayberger C, Krensky AM, Norment AM, Littman DR, Parham P.A binding site for the T-cell co-receptor CD8 on the alpha 3 domain of HLA-A2. Nature. 1990, 345(6270): 41-46.
[49] Gumperz JE, Litwin V, Phillips JH, Lanier LL, Parham P. The Bw4 public epitope of HLA-B molecules confers reactivity with natural killer cell clones that express NKB1, a putative HLA receptor. J Exp Med. 1995, 181(3): 1133-1144.
[50] Colonna M, Borsellino G, Falco M, Ferrara GB, Strominger JL. HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells. Proc Natl Acad Sci U S A. 1993, 90(24): 12000-12004.
[51] Biassoni R, Falco M, Cambiaggi A, Costa P, Verdiani S, Pende D, Conte R, Di Donato C, Parham P, Moretta L. Amino acid substitutions can influence the natural killer
(NK)-mediated recognition of HLA-C molecules. Role of serine-77 and lysine-80 in the target cell protection from lysis mediated by "group 2" or "group 1" NK clones. J Exp Med. 1995, 182(2): 605-609.
[52] Cella M, Longo A, Ferrara GB, Strominger JL, Colonna M. NK3-specific natural killer cells are selectively inhibited by Bw4-positive HLA alleles with isoleucine 80. J Exp Med. 1994, 180(4): 1235-1242.
[53] Gaubin M, Houlgatte R, Dettin M, et al. Definition of the alpha 2 region of HLA-DR molecules involved in CD4 binding. Hum Immunol 1999, 60(4): 273-281.
[54] Soin B, Vial CM, Friend PJ. Xenotransplantation. British Journal of Surgery, 2000, 87(2): 138-148.
[55] Brunsberg U, Edfors-Lilja I, Andersson L, Gustafsson K. Structure and organization of pig MHC class Ⅱ DRB genes: evidence for genetic exchange between loci. Immunogenetics, 1996, 44(1): 1-8.
[56] Kanai TH, Tanioka Y, Tanigawa M, Matsumoto Y, Ueda S, Onodera T, Matsumoto Y. Allelic diversity at class Ⅱ DRB 1 and DQB loci of the pig MHC (SLA). Immtmogenetics. 1999, 50(5-6): 295-300.
[57] Chen FX, Tang J, Li NL, Shen BH, Zhou Y, Xie J, Chou KY. Novel SLA class Ⅰ alleles of Chinese pig strains and their significance in xenotransplantation. Cell Res. 2003, 13(4): 285-294.
[58] Xia G, Ji P, Rutgeerts O, Waer M. Natural killer cell- and macrophage mediated discordant guinea pig--＞rat xenograft rejection in the absence of complement, xenoantibody and T cell immunity. Transplantation. 2000, 70(1): 86-93.
[59] Ortaldo JR, Winkler-Pickett R, Willette-Brown J., et al. Structure/function relationship of activating Ly-49D and inhibitory Ly-49G2 NK receptors. Immunol 1999, 163 (10): 5269-5277.
[60] Forte P, Pazmany L, Matter-Reissmann UB, Stussi G, Schneider MK, Seebach JD. HLA-G inhibits rolling adhesion of activated human NK cells on porcine endothelial cells. J Immunol, 2001, 167(10): 6002-6008.
[61] Platt JL. The immunological hurdles to cardiac xenotransplantation. J Card Surg. 2001, 16(6): 439-447.
[62] MacKenzie DA, Huller DA, Sollinger HW. Xenogeneic transplantation of porcine islets: an overview. Transplantation. 2003, 76(6): 887-891.
[63] Waddell TK, Peterson MD. Lung transplantation. Xenotransplantation. Chest Surg Clin N Am. 2003, 13(3): 559-576.
[64] Rayat GR, Gill RG. Pancreatic islet xenotransplantation: barriers and prospects. Curr Diab Rep. 2003, 3(4): 336-343.
[65] Hammerman MR. Xenotransplantation of developing kidneys. Am J Physiol Renal Physiol. 2002, 283(4): F601-606.
[66] Brevig T, Holgersson J, Widner H. Xenotransplantation for CNS repair: immunological barriers and strategies to overcome them. Trends Neurosci. 2000, 23(8): 337-344.
[67] Niemann H, Kues WA. Application of transgenesis in livestock for agriculture and biomedicine. Anim Reprod Sci. 2003, 79(3-4): 291-317.
[68] Platt JL. Genetic modification of xenografts. Curr Top Microbiol Immunol. 2003, 278: 1-21.
[69] Denning C, Priddle H. New frontiers in gene targeting and cloning: success, application and challenges in domestic animals and human embryonic stem cells. Reproduction. 2003, 126(1): 1-11.
[70] Splendiani G, Cipriani S, Vega A, Casciani CU. Artificial organs and transplantation. Artif Cells Blood Substit Immobil Biotechnol. 2003, 31 (2): 91-96.
[71] Lai L, Prather RS. Progress in producing knockout models for xenotransplantation by nuclear transfer. Ann Med. 2002, 34(7-8): 501-506.
[72] Machaty Z, Bondioli KR, Ramsoondar JJ, Fodor WL. The use of nuclear transfer to produce transgenic pigs. Cloning Stem Cells. 2002, 4(1): 21-27.
[73] Polejaeva IA. Cloning pigs: advances and applications. Reprod Suppl. 2001, 58: 293-300.
[74] Sandrin MS, Loveland BE, McKenzie IF. Genetic engineering for xenotransplantation. J Card Surg. 2001, 16(6): 448-457.
[75] Cascalho M, Platt JL. Xenotransplantation and other means of organ replacement. Nat Rev Immunol. 2001, 1(2): 154-160.
[76] Ohdan H, Sykes M. B cell tolerance to xenoantigens. Xenotransplantation. 2003, 10(2):
98-106.
[77] Knosalla C, Cooper DK. Xenotransplantation and tolerance. Front Biosci. 2002, 1(7): d1280-1287.
[78] Mollnes TE, Fiane AE. Perspectives on complement in xenotransplantation. Mol Immunol. 2003, 40(2-4): 135-143.
[79] Candinas D, Bach FH, Hancock WW. Delayed xenograft rejection in complement-depleted T-cell-deficient rat recipients of guinea pig cardiac grafts. Transplant Proc, 1996, 28(2): 678
[80] Lin Y, Soares MP, Sato K., et al. Rejection of cardiac xenografts by CD4+ or CD8+ T cells. Immunol, 1999, 162 (2): 1206-1214.
[81] 龚非力。医学免疫学，2000，人民卫生出版社，第75-76页。
[82] 陈慰峰。医学免疫学，2000，第三版，人民卫生出版社，第84-85页。
[83] 周光炎。免疫学原理，2000，上海科学技术文献出版社，第187-188页。
[84] Hanson P, Heuser J E, Jahn R. Neurotransmitter release—four years of SNARE complexes. Current Opinion in Neurobiology, 1997, 7: 310-315.
[85] Hay JC. SNARE Complex Structure and Function. Experimental Cell Research, 2001, 271: 10-12.
[86] Guo Z, Turner C, Castle D. Relocation of the t-SNARE SNAP-23 from lamellipodia-like cell surface projections regulates compound exocytosis in mast cells. Cell, 1998, 94(4): 537-548.
[87] Chen D, Bernstein A M, Lemons P P. et al. Molecular mechanisms ofplatelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release. Blood, 2000, 95(3): 921-929.
[88] Hibi T, Hirashima N, Nakanishi M. Rat basophilic leukemia cells express syntaxin-3 and VAMP-7 in granule membranes. Biochemical and Biophysical Research communications, 2000, 271(1): 36-41.
[89] Paumet F, Mao J L, Martin S, et al. soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment. J. Immunology, 2000, 164(11): 5850-5857.
[90] Drexler HG and Matsuo Y. Malignant hematopoietic cell lines: in vitro models for the study of natural killercell leukemia-lymphoma. Leukemia, 2000, 14(5): 777-782.
[91] Tonn T, Beckr S, Esser R, et al. Celluar immunotherapy of malignancies using the clonal natural killer cell line NK-92. J. Hematother Stem Cell Res, 2001, 10(4): 535-544.
[92] Sollner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, Tempst P and Rothman JE. SNAP receptors implicated in vesicle targeting and fusion. Nature, 1993, 362(6418): 318-324.
[93] Sutton RB, Fasshauer D, Jahn R and Brunger AT. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4A resolution. Nature, 1998, 395(6700): 347-353.
[94] 吴颖等。神经胞吐分子机制的研究进展。中国神经科学杂志，2000，16(3)：272-275．
[95] Ravichandran, V., Chawla, A., and Roche, P. A. Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues. J. Biol. Chem., 1996, 271(23): 13300-13303.
[96] Schulz, J. R., Sasaki, J. D., and Vacquier, V. D. Increased association of synaptosome-associated protein of 25 kDa with syntaxin and vesicle-associated membrane protein following acrosomal exocytosis of sea urchin sperm. J. Biol. Chem., 1998, 273 (38): 24355-24359.
[97] Shukla A, Cordon T J, Nielsen S, et al. Biochemical and Biophysical Research Communications, 2001, 285(2): 320-327.
[98] Martin B M, Nabokina S M, Blasi J, et al. Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis, Blood, 2000, 96(7): 2574-2583.
[99] Leung S M, Chen D, DasGupta B R, et al. SNAP-23 requirement for transferring recycling in Streptolysin-O-permeabilized Madin-Darby canine kidey cells. J. Biol. Chem., 1998, 273 (28): 17732-17741.
[100] Rea S, Martin L B, McIntosh S, et al. An adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface. J. Biol. Chem., 1998, 273(30): 18784-18792.
[101] Xiang Z. and Nilsson G. IgE receptor-mediated release of nerve growth factor by mast cells. Clinical and Experimental Allergy, 2000, 30 (10): 1379-1386.
[102] Chen D, Lemons PP, Schraw T. et al. Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 and 4 in lysosome release. Blood, 2000, 96 (5): 1782-1788.
[103] Hoffmann A, Jamin A, Foetisch K, May S, Aulepp H, Haustein D and Vieths. Determination of the allergenic activity of birch pollen and apple prick test solutions by measurement of β-hexominidase release from RB-2H3cells (Comparison with classical methods in allergen standardization). Allergy, 1999, 54(5): 446-454.
[104] Bonnema JD, Rivlin KA, Ting AT, Schoon RA, Abraham RT, Leibson PJ. Cytokine-enhanced NK cell-mediated cytotoxicity. Positive modulatory effects of IL-2 and IL-12 on stimulus-dependent granule exocytosis. J Immunol. 1994, 152(5): 2098-2104.
[105] Ting AT, Schoon R.A, Abraham RT, Leibson PJ. Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis. J Biol Chem. 1992, 267(33): 23957-23962.
[106] 李爱玲，孙纳等。NK细胞系细胞毒效应MTT比色法优化条件探讨。《中国免疫学杂志》，2002年，第10期，第687-689页。
[107] 何金生，李瑞珠等。MTT还原法检测NK细胞活性的方法学研究。《中国免疫学杂志》，1996年，第6期，第356-358页。
[108] Richmond JE and Broadie KS. The synaptic vesicle cycle: exocytosis and endocytosis in Drosophila and C. elegans. Current Opinion in Neurobiology. 2002, 12(5): 499-507.
[109] Stinchcombe JC and Griffiths GM. Regulated Secretion from Hemopoietic Cells. J Biol Chem.1999, 147(1): 1-6
[110] Liu JH, Wei S, Blanchard DK, Djeu JY. Restoration of lytic function in a human natural killer cell line by gene transfection. Cell Immunol. 1994, 156(1): 24-35.
[111] Miller JS, Tessmer-Tuck J, Blake N, Lund J, Scott A, Blazar BR, Orchard PJ. Endogenous IL-2 production by natural killer cells maintains cytotoxic and proliferative capacity following retroviral-mediated gene transfer. Exp Hematol. 1997, 25(11): 1140-1148.
[112] Nagashima S, Mailliard R, Kashii Y, Reichert TE, Herberman RB, Robbins P, Whiteside TL. Stable transduction of the interleukin-2 gene into human natural killer
cell lines and their phenotypic and functional characterization in vitro and in vivo. Blood. 1998, 91(10): 3850-3861.
[113] Tam YK, Maki G, Miyagawa B, Hennemann B, Tonn T, Klingemann HG. Characterization of genetically altered, interleukin 2-independent natural killer cell lines suitable for adoptive cellular immunotherapy. Hum Gene Ther. 1999, 10(8): 1359-1373.
[114] Trompeter HI, Weinhold S, Thiel C, Wemet P, Uhrberg M. Rapid and highly efficient gene transfer into natural killer cells by nucleofection. J Immunol Methods. 2003, 274(1-2): 245-256.
[115] Jeffrey SM. The biology of natural killer cells in cancer, infection, and pregnancy. Exp. Hematology. 2001, 29(10): 1157-1168.
[116] Flodstrom M, Shi FD, Sarvetnick N et al. The natural killer cell—friend or foe in autoimmune disease? Stand J. Immunol. 2002, 55(5): 432-441.