实验性抗磷脂抗体综合征小鼠CD4~+CD25~+调节性细胞及Foxp3表达的实验研究
|
摘要
抗磷脂抗体综合征(Antiphospholipid antibody syndrome, APS)是一种以习惯性流产、动静脉血栓形成为主要特征的一类累及多器官、系统的自身免疫病,患者血中存在抗磷脂抗体(Antiphospholipid antibody, APA)。现已知β2糖蛋白1(β2-glycoprotein-1,β2-GP1)是高度糖基化的单链血浆蛋白,是APA (anti-β2GP I)主要的靶抗原。大量证据显示抗β2糖蛋白1抗体(anti-beta2-glycoprotein-1 antibody, anti-β2GP I)、抗心磷脂抗体(anticardiolipin antibodies, ACA)以致炎因子生成、内皮细胞和血小板活化、凝血因子和补体激活等机制参与APS的发病。CD4+CD25+调节性T细胞(CD4+CD25+regulatory T cell, Treg)是调节性T细胞中最为重要的一群,因其具有免疫调节和抑制作用,近年来受到研究者的密切关注。研究表明调节性T细胞对于维持机体免疫自稳,防止自身免疫病具有极其重要的作用,资料显示CD4+CD25+Treg的数量减少或功能异常可与多种自身免疫病相关。但对于在APS及APS实验动物模型中CD4+CD25+Treg功能的变化国内外却鲜见报道。
本实验将以重组人p2糖蛋白1(recombinant humanβ2-glycoprotein-1, rhβ2GP I)主动免疫BALB／c小鼠建立实验性APS模型,采用流式细胞术检测小鼠PBMC中CD4+CD25+Treg细胞数目,RT-PCR检测转录因子Foxp3 mRNA的表达,以观察CD4+CD25+Treg细胞数目变化及活性变化是否参与APS的发病机制。结果发现,在应用rhβ2GPⅠ免疫小鼠12w后,CD4+CD25+Treg细胞亚群的比例及Foxp3 mRNA表达开始明显降低,这与rhβ2GPⅠ免疫小鼠开始出现APS疾病的临床征象时间一致。提示CD4+CD25+Treg细胞在机体内起重要的免疫调节作用,正常情况下它与效应性T细胞互相拮抗抑制,随着CD4+CD25+Treg细胞数量及活性下降后抑制作用减弱,表现出效应性T细胞发育旺盛从而表现出APS的临床征象。CD4+CD25+Treg细胞数量的多少及活性的高低是发生APS的重要发病因素。我们期待CD4+CD25+Treg细胞数量的多少及活性的高低能否成为日后诊断APS的辅助诊断依据,通过监测CD4+CD25+Treg细胞数量的多少及活性的高低能否成为预防APS的检验手段,以及患有APS疾病的人们出现CD4+CD25+Treg细胞数量的减少及活性的降低,输注CD4+CD25+Treg细胞能否成为有效的治疗手段。随着对APS发病机制的深入研究,人类将找到有效的检测及治疗方法。
Objective:To observe the quantitative and functional changes of CD4+CD25+regulatory cells in mice during the development of experimental anti-phospholipid antibody syndrome (EAPS). Methods:To establish the model of EAPS, we immunized BALB/c mice with rhβ2-GP1. Twelve weeks after immunization, aCL、anti-β2GPI titers in the sera, the percentage of the fetal resorptions, activated partial thromboplastin time (aPTT) and platelet counts were assayed. Frequency of CD4+CD25+Treg in peripheral blood mononuclear cells from EAPS mice were determined by flow cytometry, mRNA expression of foxp3 were semi-quantified by RT-PCR. Results:Model mice showed an elevated titers of anti-β2GPI and aCL Abs in the sera (p<0.05), higher resorption percentage (p<0.05), prolonged aPTT (p<0.05), and lower platelet counts (p<0.05). The expressing levels of foxp3 mRNA in the model group was higher than those of control group (p<0.05) in the fourth week after immunization, but the expressing level of Foxp3 mRNA began to decrease in week 8 and were lower than those of control group from week twelve (p<0.05). The frequency of CD4+CD25+Treg cells in model group was similar with that of control group within the eighth week after immunization (p>0.05), but after week twelve, the frequency of CD4+CD25+Treg cells in model group began to get lower than contol group (p<0.05).
Conclusion:The decreased number and function of CD4+CD25+Treg cells may contribute to the pathogenic mechanism in EAPS.
本实验将以重组人p2糖蛋白1(recombinant humanβ2-glycoprotein-1, rhβ2GP I)主动免疫BALB／c小鼠建立实验性APS模型,采用流式细胞术检测小鼠PBMC中CD4+CD25+Treg细胞数目,RT-PCR检测转录因子Foxp3 mRNA的表达,以观察CD4+CD25+Treg细胞数目变化及活性变化是否参与APS的发病机制。结果发现,在应用rhβ2GPⅠ免疫小鼠12w后,CD4+CD25+Treg细胞亚群的比例及Foxp3 mRNA表达开始明显降低,这与rhβ2GPⅠ免疫小鼠开始出现APS疾病的临床征象时间一致。提示CD4+CD25+Treg细胞在机体内起重要的免疫调节作用,正常情况下它与效应性T细胞互相拮抗抑制,随着CD4+CD25+Treg细胞数量及活性下降后抑制作用减弱,表现出效应性T细胞发育旺盛从而表现出APS的临床征象。CD4+CD25+Treg细胞数量的多少及活性的高低是发生APS的重要发病因素。我们期待CD4+CD25+Treg细胞数量的多少及活性的高低能否成为日后诊断APS的辅助诊断依据,通过监测CD4+CD25+Treg细胞数量的多少及活性的高低能否成为预防APS的检验手段,以及患有APS疾病的人们出现CD4+CD25+Treg细胞数量的减少及活性的降低,输注CD4+CD25+Treg细胞能否成为有效的治疗手段。随着对APS发病机制的深入研究,人类将找到有效的检测及治疗方法。
Objective:To observe the quantitative and functional changes of CD4+CD25+regulatory cells in mice during the development of experimental anti-phospholipid antibody syndrome (EAPS). Methods:To establish the model of EAPS, we immunized BALB/c mice with rhβ2-GP1. Twelve weeks after immunization, aCL、anti-β2GPI titers in the sera, the percentage of the fetal resorptions, activated partial thromboplastin time (aPTT) and platelet counts were assayed. Frequency of CD4+CD25+Treg in peripheral blood mononuclear cells from EAPS mice were determined by flow cytometry, mRNA expression of foxp3 were semi-quantified by RT-PCR. Results:Model mice showed an elevated titers of anti-β2GPI and aCL Abs in the sera (p<0.05), higher resorption percentage (p<0.05), prolonged aPTT (p<0.05), and lower platelet counts (p<0.05). The expressing levels of foxp3 mRNA in the model group was higher than those of control group (p<0.05) in the fourth week after immunization, but the expressing level of Foxp3 mRNA began to decrease in week 8 and were lower than those of control group from week twelve (p<0.05). The frequency of CD4+CD25+Treg cells in model group was similar with that of control group within the eighth week after immunization (p>0.05), but after week twelve, the frequency of CD4+CD25+Treg cells in model group began to get lower than contol group (p<0.05).
Conclusion:The decreased number and function of CD4+CD25+Treg cells may contribute to the pathogenic mechanism in EAPS.
引文
[1]George D, Erkan D. Antiphospholipid syndrome. Prog Cardiovasc Dis.2009,52(2): 115-25.
[2]Levine JS, Branch DW, Rauch J. The antiphospholipid syndrone. N Engl J Med,2002, 346:752-763.
[3]Merrilll JT. Antibodies and clinical features of the antiphospholipid syndrome as criteria for systemic lupus erythematosus. Lupus,2004,13:869-876
[4]Roubey RAS. Autoantibodies to phospholipid-binding plasima proteins:a new view of lupus anticagulants and other antiphospholipid autoantibodies. Blood 1994,84:2854-2867.
[5]Roubey R. Zmmunology of the auti phospholipid syndnome Arthirtis Rheum 1996, 39:1444-1454
[6]Pierangeli SS, Chen PP, Raschi E, et al. Antiphospholipid antibodies and the antiphospholipid syndrome:pathogenic mechanisms. Semin Thromb Hemost.2008, 34(3):236-50.
[7]Schultze H.E, Heide K, Haupt H. Uber einbisher unbekanntes niedermole kulares β 2-globulin des human serums. Naturwissenschaften,1961,48:791.
[8]McNeil HP, Simpson RJ, Chesterman CN, et al. Antiphospholipid antibodies are directed against a complexantigen that includes a lipid-binding inhibitor ofcoagulation: b-glycoprotein I (apolipoprotein H). Proc 2Natl Acad Sci USA 1990,87:4120-4.
[9]Sarmarco M, Soler C, Christides C. Prevalence and clinical significance of IgG isotope nati-beta2-glycoprotein 1 antibodies in anti-phospholipid syndrome[J]. J Lab Clin,1997, 129(5):499-506.
[10]Andre S, Tough DF, Lacroix-Desmazes S, et al. Am J Pathol. Surveillance of antigen-presenting cells by CD4+CD25+regulatory T cells in autoimmunity: immunopathogenesis and therapeutic implications.2009,174(5):1575-87.
[11]Mu L, Sun B, Kong Q, et al. Disequilibrium of T helper type 1,2 and 17 cells and regulatory T cells during the development of experimental autoimmune myasthenia gravis. Immunology.2009,128(1 Suppl):826-36.
[12]Yang J, Chu Y, Yang X, et al. Th17 and natural Treg cell population dynamics in systemic lupus erythematosus. Arthritis Rheum.2009 May,60(5):1472-83.
[13]Wassermann A, Neisser A, Bruck C. Eine serodiagnostische reaction bei syphilis [J]. Dtsch Med Wochenschr,1906,32:745-746.
[14]Lolg AA, Ginsberg Js, Brill-Edwards P, et al. The relationship of antiphospholipid abtibodies to thromboembolic disease in systemic lupus erythematomus:a cross-sectional study [J]. Thromb Haemost,1991,66(5):520-524.
[15]Gamia-Torres R, Amigo MC, De la Rosa A et al.Iupus,1996,5:56-61.
[16]Moore JE, Mohr CF. Biologically false positive serological tests for syphilis:type, incidence, and cause. J Am Med Assoc,1952,150:467-473.
[17]Moore JE, Lutz WB. Natural history of systemic lupus erythematosus:approach to its study through chronic biologic false positive reactors. J Chronic Dis.1955,1:297-316.
[18]Shapiro SS, Thiagarajan P. Lupus anticoagulants. Prog Hemost Thromb.1982,6: 263-285.
[19]Conley CL, Hartmann RC. A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest.1952,31:621.
[20]Feinstein DI, Rapaport SI. Acquired inhibitors of blood coagulation. In:Spaet TH, ed. Progress in Hemostasis and Thrombosis. Vol 1. New York, NY:Grune& Stratton; 1972:75-95.
[21]Bowie WEJ, Thompson JH, Pascuzzi CA, et al. Thrombosis in systemic erythematosus despite circulating anticoagulants. J Clin Invest.1963,62:416-430.
[22]Laurell A, Nilsson I. Hypergammaglobulinemia, circulating anticoagulant and biologic false-positive Wassermann reaction. J Lab Clin Med.1957,49:694-707.
[23]Harris EN, Gharavi AE, Boey ML, et al. Anticardiolipin antibodies:detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet.1983,2:1211-1214.
[24]Hughes GR. The anticardiolipin syndrome. Clin Exp Rheumatol.1985,3:285-286.
[25]Harris EN, Hughes GRV, Gharavi AE. Antiphospholipid antibodies:an elderly statesman dons new garments. J Rheumatol.1987,14(suppl 13):208-213.
[26]Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the calssification criteria for definite antiphospholipid syndrome(APS)[J]. J Thromb Haemost,2006,4(2):295-306.
[27]Hughes GRV. Postgraduate Medical Journal 1988,64:517.
[28]Gamia-Torres R, Amigo MC, De la Rosa A, et al.lupus,1996,5:56-61.
[29]Sandoval J, ergo MC, Barragan R et al. J Rheumatol,1996,23:772-775.
[30]Abu-Shakra M, Urowitz MB, Gladman DD et al. Lupus,1996,5:70-73.
[31]Guibal F, Rybojad M, Cordoliaru F, et al. Dermatology,1996,192:75-77.
[32]Blank M, Krause I, Fridinkin M, et al. Bacterial induclion of autoantibodies to betel-glycoprotein-I accounts for the infectious etiology of antiphnspholipid sgndromme. J Clin Cnveat,2002,109:797-804.
[33]Gharavi AE, Pierangeli SS, Espinulu RG, et al. Antiphospholipid antibodies induces in mice by immunixation with a cytomegalovirus-derived Peptide cause Tbromhnsis aad ac Tiva Tian of endothelial cells in vivo. Arthritis Rheum,2002,46:545-552.
[34]Arnout J, Yerrnylen J. Currant status and implications of autoimmunc antiphospholipid antibodies in relation to thrombotic disease. J Thromb Haemost,2003,1:931-942.
[35]Robertson B, GteavexM.3ntiphnspholipid syndrome:an evolving story. Blood Rev,2006, 20:201-212.
[36]Pierangeli SS, Chen PP, Gonzblez EB. Antiphospholipid antibodies and the antiplruspholipid syndrome:an update on trealrnrnt and pathogenic mechanisms. Cure Opin Hcmatol,2006,13:366-375.
[37]Price Y, Reddel SW, Herzog H, et al. Impaired thrombin generation in B2-glycoprotein I null mice. J Biol Chem.2001,276:13817-13821.
[38]Gushiken FC, Arnett FC, Thiagarajan P. Primary antiphospholipid antibody syndrome with mutations in the phospholipid binding domain of B2-glycoprotein I. Am J Hematol. 2000,65:160-165.
[39]Garcia CO, Kanbour-Shakir A, Tang H, et al. Induction of experimental antiphospholipid antibody syndrome in PL/J mice following immunization with B2 GPI. Am J Reprod Immunol.1997,37:118-124.
[40]Blank M, Faden D, Tincani A, et al. Immunization with anticardiolipin cofactor (B-2-glycoprotein I) induces experimental antiphospholipid syndrome in naive mice. J Autoimmun.1994,7:441-455.
[41]Pierangeli SS, Harris EN. In vivo models of thrombosis for the antiphospholipid syndrome. Lupus.1996,5:451-455.
[42]Pierangeli SS, Barker JH, Stikovac D, et al. Effect of human IgG antiphospholipid antibodies on an in vivo thrombosis model in mice. Thromb Haemost.1994,71:670-674.
[43]Olee T, Pierangeli SS, Handley HH, et al. A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice. Proc Natl Acad Sci USA.1996,93:8606-8611.
[44]Ames PR, Tommasino C, Iannaccone L, et al. Coagulation activation and fibrinolytic imbalance in subjects with idiopathic antiphospholipid antibodies-a crucial role for acquired free protein S deficiency. Thromb Haemost.1996,76:190-194.
[45]Schousboe I, Rasmussen MS. Synchronized inhibition of the phospholipid mediated autoactivation of factor XII in plasma by B2-glycoprotein I and anti-Β2-glycoprotein I. Thromb Haemost.1995,73:798-804.
[46]Luong T-H, Rand JH, Wu XX, et al. Seasonal distribution of antiphospholipid antibodies. Stroke.2001,32:1707-1711.
[47]Shibata S, Harpel PC, Gharavi A, et al. Autoantibodies to heparin from patients with antiphospholipid antibody syndrome inhibit formation of antithrombin III-thrombin complexes. Blood.1994,83:2532-2540.
[48]Kamboh, ML, Ferrell. RE and Sepehmia B. Genetic studies of human apolipoprotein. IV. Structural heterogeneity of apolipoprotein H. Am. J. Hum. Genet.1988,4(3):452-457.
[49]Gries A, Nimpf j, Wurm H, et al. Characterization of isoelelctric subspecies of asialo-beta 2-GP Ⅰ. Biochem j.1989,260:531-534.
[50]Sheng Y, Reddel SW, Herzog H, et al. Impaired thrombin generation in B2-glycoprotein I null mice. J Biol Chem.2001,276:13817-13821.
[51]Chanley LW, Allen JL, Johnson PM. Synthesis of beta2-glycotrotein I by the human placenta. Placenta.1997,18:403-410.
[52]McNeil HP, Simpson RJ, Chesterman CN, et al. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid binding inhibitor of coagulation: beta-2 Glycoprotein-1. Proceed Natl Acad Sci USA.1990,87:4120-4124.
[53]Galli M, Comfurius P, Maassen C, et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990,335:1544-1547.
[54]Matsuura E, Igarashi Y, Eujimoto, M., et al. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990,336:177-178.
[55]Lozier J, Takahashi N, Putnam FW, et al. Complete amino acid sequence of human plasma beta 2-gylcoprotein I. Proc Natl Acad Sci USA.1984,81:3640-4.
[56]PG. DE GROOT, RHWMD ERKSEN. Pathophysiology of antiphoepholipid antibodies [J]. The Netherlands journal of medicine,2004,62(8):267-272.
[57]JANINE J.J. HUISTEIN, PETER J, et al. Beta2-Glycoprotein I inhibits von Willehrand factor-dependent platelet adhesion and aggregation [J]. Blood,2007,110(5):1483-14910.
[58]YASUDA S, ATSUMI T, IEKO M, et al. Nicked beta 2-glycoprotein Ⅰ;a marker of cerebral infarct end a novel role in the negative feedLaek pathway of extrinsic fibrinolysis [J]. Blood,2004,103(10):3766-3772.
[59]Sttinkasserer T, Estaller C, Weiss EH, et al. Complete nucleotide and deduced amino acid sequence of human B2-glycoporotein 1. Biochem J 1991,277:387-391.
[60]Arrieux J, Roussel B, Jacob MC, et al. Measurement of antiphospholipol antibodies by ELISA using B2-ghycoprotein as an antigen J Immunol Methods 1991,143:223-229.
[61]Cabral AR, Alar con-stgovia D. Anti-beta 2 gly coprotein! Antibody testing in pationts with amtiphosphlipid syndome. Br J Rhoumato! 1997,36(11):1235.
[62]Tsutsumi A, Matsuura E, IchiKawa K, et al. IgA dass anti-beta-2-ghycoprotein in pationts with systemic lupus erythemtosus J khenmatol,1998,25(1):74-78.
[63]Kamboh, ML, Ferrell. RE and Sepehmia B. Genetic studies of human apolipoprotein. IV. Structural heterogeneity of apolipoprotein H. Am. J. Hum. Genet.1988,4(3):452-457.
[64]Haginara Y, Hong DP, Hoshino M, et al. Aggregation of beta2-glycoprotein I induced by sodium lauryl suffate and lysophos pholipids Buicgenstrt 2002 Jan 341(3): 1020-1026.
[65]Hunt J and Krilis S. The fifth domain of beta 2-glycoprotein I contains a phosphlipid binding site (Cys281-Cys288) and a region recognized by anticardiolipin antibodies. J Immunol.1994,152:653-659.
[66]Porter TF. Antiphospholipid antibodies and infertility. Clin Obstet Gynecol.2001,44: 29-35.
[67]Schwarzenbacher R, Zeth K, Diederichs K, et al. Crystal structure of human Β2-glycoprotein I:implications for phospholipid binding and the antiphospholipid syndrome. EMBO J.1999,18:6228-6239.
[68]Wurm H, Beubler E, Polz E. Studies on the possible function of beta2-glycoprotein 1: influence in the triglyceride metabolism in the rat Metabolism.1982,31(5):484-486.
[69]Brighton AT, Hogg PJ, Dai YP, et al. Beta-2-gylcoprotein I in thrombosis:evidence for a role as a natural anticoagulant. Br J Haemato,1996,93(1):185-194.
[70]Chonn A, Semple SC, Cullis PR. Beta-2-glycoprotein I is a major protein associated with very rapidly cheated? Liposomes in vivo, suggesting a significant role in the immune clearance of "non-self" particles. J Boil Chem.1995,270:25854-25859.
[71]Horhach D A, van Oort E, Donders K C, et al. Lupus amticoaguhnrt is the strongest risk factor for both venous amd arterial thrombosis in patients with svsteanic lupus ervtheanatosus. Comparison hetwexn different assays for the detection of mrtiphospholipid mrtibodies. Thromh Haemost,1996,76(6):916.
[72]Reher G, Arvieux J, Comby E, et al. Multicenter evaluation of nine commercial kits for the qumititation of amticnrdiolipin amtihodies. Thromh Haemost,1995,73(3):444.
[73]Me Neil HP, Smpson RJ, Chestenmm CN, et al. Antiphospholipid antibodies are directed agangainstnst a complex antigen that includes a lipid binding inhibitor of coagulation:β 2 Glycoprotein I (apolipoprotein H). Proc Natl Acad Sci LSA,1990, 87(11):4120.
[74]Tincan A, Spatola L, Balestrieri G. β 2GPI cofactor and ACA. Isr, J Med Sci,1995, 31(1):17.
[75]George J, Shoenfeld Y, Harats D. The involvement of beta 2-glycoprotein 1 (beta2-GP1) in human and murine atherosclerosis. J Autoimmun,1999,13:57-60.
[76]Finazzi G. The epidemiology of the antiphospholipid syndrome:who is at risk Curr Rheumatol Rep,2001,3:271-276.
[77]临床免疫学和免疫检验／王兰兰主编-北京-科学技术文献出版社,2004.10.
[78]P.G.DE GROOT, R.H.W.M. DERKSEN. Pathophysiology ofan-tiphospholipid antibodies [J]. The Netherlands journal of medicine,2004,62(8):267-272.
[79]JANINE J J. HULSTEIN, PETER J, et al. Beta2. Glycoprotein linhibits von Willebmnd factor-dependent platelet adhesion and aggregation[J]. Blood,2007,110(5):1483-1491.
[80]YASUDA S, ATSUMI T, IEKO M, et al. Nicked beta 2-glycoproteinI:a marker of 333 cerebral infarct and a novel role in the negativefeedba Sarmarco M, Soler C, Christides C.Prevalence and clinical significance of IgG isotope nati-beta2-glycoprotein 1 antibodies in anti-phospholipid syndrome[J]. J Lab Clin,1997,129(5):499-506.
[81]医学免疫学／金伯泉主编／第5版.人民卫生出版社,2008.6.
[82]Sakaguchi S.Naturally arising CD4+regulatory T cells for immunologic self-toleracnce and negative control of immune responses[J]. Annu Rev Immunol,2004,22:531.
[83]Shevach, EM. Regulatory T cells in autoimmunity[J]. annu Rev Immunol,2000,18:423.
[84]Picca CC, Caton AJ. The role of self-peptides in the development of CD4+CD25+ regulatory T cells[J]. Curr Opin Immunol,2005,17(4):131-136.
[85]付嘉,方艳秋,徐立,等.人β2糖蛋白1的重组表达及对抗磷脂抗体综合征患者血清诱导内皮细胞产生ICAM-1的影响[J].吉林大学学报(医学版),2006,32(4)：654-657.
[86]Angela T, Flavio A, Genesio B, et al. Minimal requirements for antiphospholipid antibodies ELISA proposed by the European Forum on antiphospholipid sntibodies[J]. Thrombosis Research,2004,114:553-558.
[87]Tolomeo T, Rico De Souza A, Roter E, et al. T cells demonstrate a Thl-biased response to native beta2-glycoprotein I in a murine model of anti-phospholipid antibody induction. Autoimmunity.2009,42(4):292-5.
[88]Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases[J]. Immunol,1995, 155(3):1151-1164.
[89]Banham AH. Cell-surface IL-7 receptor expression facilitates the purification of Foxp3+ regulatory T cells [J]. Trends Imm unol,2006,27(12):541-544.
[90]Corthay A. How do regulatory T cells work Scand J Immunol.2009 Oct, 70(4):326-36.
[91]孟荔,欧阳建.CD4+CD25+调节性T细胞与自身免疫病.中国组织工程研究与临床康复,2007,11(33)：6676-6680.
[92]D. M. Waid, G. M. Vaitait is, N. D. Pennock, et al. Disruption of the homeostatic balance between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes diabetes. J. Leukoc. Biol. August 1,2008,84(2):431-439.
[93]Kuhn A, Beissert S, Krammer PH. CD4+CD25+regulatory T cells in human lupus erythematosus. Arch Dermatol Res.2009 Jan,301(1):71-81.
[94]Moldigilin Wan S, Xia C, Morel L. IL-6 p roduced by dendritic cells from lupus-prone miceinhibits CD4+CD25+T cell regulatory functions[J]. J Imm unol,2007,178(1):271-279.
[95]Valencia X, Stephens G, Goldbach Mansky R, et al. TNF down-modulates the function of human CD4+CD25hi T regulatory cells[J]. Blood,2006,108(1):253-261.
[96]Pillai V, Ortega SB, Wang CK, et al. Transient regulatory T cells:A state attained By all activeated human T cells[J]. Clin Immumol,2007,123:18-29.
[97]Sakaguchi S. Naturally arisiy CD4+regulatory T cells for immunologic self-tolerance and negative control of immune responses [J]. Annu Rev Immunol,2004,22:531-562.
[98]Construction and characterization of a novel DNA vaccine that is potent antigen-specific tolerizing therapy for experimental arthritis by increasing CD4+CD25+Treg cells and inducing Thl to Th2 shift in both cells and cytokine(song X, Liang F, Liu N, Luo Y, Xue H, Yuan F, Tan L, Sun Y, Xi C, Xi Y.Vaccine.2009 Jan 29,27(5):690-700. Epub 2008 Dec 16.)
[99]Zhang B, Sun C, Qu Y, et al. Deficiency of mouse CD4+CD25+Foxp3+regulatory T cells in xenogeneic pig thymus-grafted nude mice suffering from autoimmune diseases. Cell Mol Immunol.2008 Oct,5(5):325-32.
[100]Hall HT, Sjolin H, Brauner H, et al. Increased diabetes development and decreased function of CD4+CD25+Treg in the absence of a functional DAP 12 adaptor proteinEur J Immunol.2008 Nov,38(11):3191-9.
[101]Identification of correlations between numbers of CD4+CD25+Treg cells, levels of sera anti-AChR antibodies and transfer growth factor-beta in patients with myasthenia gravis (Wang HB, Zhang JB, Chui LY. Zhonghua Yi Xue Za Zhi.2008 Apr 15,88(15):1036-40. Chinese)
[102]Dysfunctional CD4+, CD25+regulatory T cells in untreated active systemic lupus erythematosus secondary to interferon-alpha-producing antigen-presenting cells (Yan B, Ye S, Chen G, Kuang M, Shen N, Chen S. Arthritis Rheum.2008 Mar,58(3):801-12.
[103]CD4+CD25+regulatory T cells in health and disease (Liu H, Leung BP.Clin Exp Pharmacol Physiol.2006 May-Jun; 33(5-6):519-24. Review)
[104]Paralysis of CD4+CD25+regulatory T cell response in chronic autoimmune encephalomyelitis (Matsumoto Y, Sakuma H, Kohyama K, Park IK. J Neuroimmunol. 2007 Jul; 187(1-2):44-54. Epub 2007 May 17.)
[2]Levine JS, Branch DW, Rauch J. The antiphospholipid syndrone. N Engl J Med,2002, 346:752-763.
[3]Merrilll JT. Antibodies and clinical features of the antiphospholipid syndrome as criteria for systemic lupus erythematosus. Lupus,2004,13:869-876
[4]Roubey RAS. Autoantibodies to phospholipid-binding plasima proteins:a new view of lupus anticagulants and other antiphospholipid autoantibodies. Blood 1994,84:2854-2867.
[5]Roubey R. Zmmunology of the auti phospholipid syndnome Arthirtis Rheum 1996, 39:1444-1454
[6]Pierangeli SS, Chen PP, Raschi E, et al. Antiphospholipid antibodies and the antiphospholipid syndrome:pathogenic mechanisms. Semin Thromb Hemost.2008, 34(3):236-50.
[7]Schultze H.E, Heide K, Haupt H. Uber einbisher unbekanntes niedermole kulares β 2-globulin des human serums. Naturwissenschaften,1961,48:791.
[8]McNeil HP, Simpson RJ, Chesterman CN, et al. Antiphospholipid antibodies are directed against a complexantigen that includes a lipid-binding inhibitor ofcoagulation: b-glycoprotein I (apolipoprotein H). Proc 2Natl Acad Sci USA 1990,87:4120-4.
[9]Sarmarco M, Soler C, Christides C. Prevalence and clinical significance of IgG isotope nati-beta2-glycoprotein 1 antibodies in anti-phospholipid syndrome[J]. J Lab Clin,1997, 129(5):499-506.
[10]Andre S, Tough DF, Lacroix-Desmazes S, et al. Am J Pathol. Surveillance of antigen-presenting cells by CD4+CD25+regulatory T cells in autoimmunity: immunopathogenesis and therapeutic implications.2009,174(5):1575-87.
[11]Mu L, Sun B, Kong Q, et al. Disequilibrium of T helper type 1,2 and 17 cells and regulatory T cells during the development of experimental autoimmune myasthenia gravis. Immunology.2009,128(1 Suppl):826-36.
[12]Yang J, Chu Y, Yang X, et al. Th17 and natural Treg cell population dynamics in systemic lupus erythematosus. Arthritis Rheum.2009 May,60(5):1472-83.
[13]Wassermann A, Neisser A, Bruck C. Eine serodiagnostische reaction bei syphilis [J]. Dtsch Med Wochenschr,1906,32:745-746.
[14]Lolg AA, Ginsberg Js, Brill-Edwards P, et al. The relationship of antiphospholipid abtibodies to thromboembolic disease in systemic lupus erythematomus:a cross-sectional study [J]. Thromb Haemost,1991,66(5):520-524.
[15]Gamia-Torres R, Amigo MC, De la Rosa A et al.Iupus,1996,5:56-61.
[16]Moore JE, Mohr CF. Biologically false positive serological tests for syphilis:type, incidence, and cause. J Am Med Assoc,1952,150:467-473.
[17]Moore JE, Lutz WB. Natural history of systemic lupus erythematosus:approach to its study through chronic biologic false positive reactors. J Chronic Dis.1955,1:297-316.
[18]Shapiro SS, Thiagarajan P. Lupus anticoagulants. Prog Hemost Thromb.1982,6: 263-285.
[19]Conley CL, Hartmann RC. A hemorrhagic disorder caused by circulating anticoagulant in patients with disseminated lupus erythematosus. J Clin Invest.1952,31:621.
[20]Feinstein DI, Rapaport SI. Acquired inhibitors of blood coagulation. In:Spaet TH, ed. Progress in Hemostasis and Thrombosis. Vol 1. New York, NY:Grune& Stratton; 1972:75-95.
[21]Bowie WEJ, Thompson JH, Pascuzzi CA, et al. Thrombosis in systemic erythematosus despite circulating anticoagulants. J Clin Invest.1963,62:416-430.
[22]Laurell A, Nilsson I. Hypergammaglobulinemia, circulating anticoagulant and biologic false-positive Wassermann reaction. J Lab Clin Med.1957,49:694-707.
[23]Harris EN, Gharavi AE, Boey ML, et al. Anticardiolipin antibodies:detection by radioimmunoassay and association with thrombosis in systemic lupus erythematosus. Lancet.1983,2:1211-1214.
[24]Hughes GR. The anticardiolipin syndrome. Clin Exp Rheumatol.1985,3:285-286.
[25]Harris EN, Hughes GRV, Gharavi AE. Antiphospholipid antibodies:an elderly statesman dons new garments. J Rheumatol.1987,14(suppl 13):208-213.
[26]Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the calssification criteria for definite antiphospholipid syndrome(APS)[J]. J Thromb Haemost,2006,4(2):295-306.
[27]Hughes GRV. Postgraduate Medical Journal 1988,64:517.
[28]Gamia-Torres R, Amigo MC, De la Rosa A, et al.lupus,1996,5:56-61.
[29]Sandoval J, ergo MC, Barragan R et al. J Rheumatol,1996,23:772-775.
[30]Abu-Shakra M, Urowitz MB, Gladman DD et al. Lupus,1996,5:70-73.
[31]Guibal F, Rybojad M, Cordoliaru F, et al. Dermatology,1996,192:75-77.
[32]Blank M, Krause I, Fridinkin M, et al. Bacterial induclion of autoantibodies to betel-glycoprotein-I accounts for the infectious etiology of antiphnspholipid sgndromme. J Clin Cnveat,2002,109:797-804.
[33]Gharavi AE, Pierangeli SS, Espinulu RG, et al. Antiphospholipid antibodies induces in mice by immunixation with a cytomegalovirus-derived Peptide cause Tbromhnsis aad ac Tiva Tian of endothelial cells in vivo. Arthritis Rheum,2002,46:545-552.
[34]Arnout J, Yerrnylen J. Currant status and implications of autoimmunc antiphospholipid antibodies in relation to thrombotic disease. J Thromb Haemost,2003,1:931-942.
[35]Robertson B, GteavexM.3ntiphnspholipid syndrome:an evolving story. Blood Rev,2006, 20:201-212.
[36]Pierangeli SS, Chen PP, Gonzblez EB. Antiphospholipid antibodies and the antiplruspholipid syndrome:an update on trealrnrnt and pathogenic mechanisms. Cure Opin Hcmatol,2006,13:366-375.
[37]Price Y, Reddel SW, Herzog H, et al. Impaired thrombin generation in B2-glycoprotein I null mice. J Biol Chem.2001,276:13817-13821.
[38]Gushiken FC, Arnett FC, Thiagarajan P. Primary antiphospholipid antibody syndrome with mutations in the phospholipid binding domain of B2-glycoprotein I. Am J Hematol. 2000,65:160-165.
[39]Garcia CO, Kanbour-Shakir A, Tang H, et al. Induction of experimental antiphospholipid antibody syndrome in PL/J mice following immunization with B2 GPI. Am J Reprod Immunol.1997,37:118-124.
[40]Blank M, Faden D, Tincani A, et al. Immunization with anticardiolipin cofactor (B-2-glycoprotein I) induces experimental antiphospholipid syndrome in naive mice. J Autoimmun.1994,7:441-455.
[41]Pierangeli SS, Harris EN. In vivo models of thrombosis for the antiphospholipid syndrome. Lupus.1996,5:451-455.
[42]Pierangeli SS, Barker JH, Stikovac D, et al. Effect of human IgG antiphospholipid antibodies on an in vivo thrombosis model in mice. Thromb Haemost.1994,71:670-674.
[43]Olee T, Pierangeli SS, Handley HH, et al. A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice. Proc Natl Acad Sci USA.1996,93:8606-8611.
[44]Ames PR, Tommasino C, Iannaccone L, et al. Coagulation activation and fibrinolytic imbalance in subjects with idiopathic antiphospholipid antibodies-a crucial role for acquired free protein S deficiency. Thromb Haemost.1996,76:190-194.
[45]Schousboe I, Rasmussen MS. Synchronized inhibition of the phospholipid mediated autoactivation of factor XII in plasma by B2-glycoprotein I and anti-Β2-glycoprotein I. Thromb Haemost.1995,73:798-804.
[46]Luong T-H, Rand JH, Wu XX, et al. Seasonal distribution of antiphospholipid antibodies. Stroke.2001,32:1707-1711.
[47]Shibata S, Harpel PC, Gharavi A, et al. Autoantibodies to heparin from patients with antiphospholipid antibody syndrome inhibit formation of antithrombin III-thrombin complexes. Blood.1994,83:2532-2540.
[48]Kamboh, ML, Ferrell. RE and Sepehmia B. Genetic studies of human apolipoprotein. IV. Structural heterogeneity of apolipoprotein H. Am. J. Hum. Genet.1988,4(3):452-457.
[49]Gries A, Nimpf j, Wurm H, et al. Characterization of isoelelctric subspecies of asialo-beta 2-GP Ⅰ. Biochem j.1989,260:531-534.
[50]Sheng Y, Reddel SW, Herzog H, et al. Impaired thrombin generation in B2-glycoprotein I null mice. J Biol Chem.2001,276:13817-13821.
[51]Chanley LW, Allen JL, Johnson PM. Synthesis of beta2-glycotrotein I by the human placenta. Placenta.1997,18:403-410.
[52]McNeil HP, Simpson RJ, Chesterman CN, et al. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid binding inhibitor of coagulation: beta-2 Glycoprotein-1. Proceed Natl Acad Sci USA.1990,87:4120-4124.
[53]Galli M, Comfurius P, Maassen C, et al. Anticardiolipin antibodies (ACA) directed not to cardiolipin but to a plasma protein cofactor. Lancet 1990,335:1544-1547.
[54]Matsuura E, Igarashi Y, Eujimoto, M., et al. Anticardiolipin cofactor(s) and differential diagnosis of autoimmune disease. Lancet 1990,336:177-178.
[55]Lozier J, Takahashi N, Putnam FW, et al. Complete amino acid sequence of human plasma beta 2-gylcoprotein I. Proc Natl Acad Sci USA.1984,81:3640-4.
[56]PG. DE GROOT, RHWMD ERKSEN. Pathophysiology of antiphoepholipid antibodies [J]. The Netherlands journal of medicine,2004,62(8):267-272.
[57]JANINE J.J. HUISTEIN, PETER J, et al. Beta2-Glycoprotein I inhibits von Willehrand factor-dependent platelet adhesion and aggregation [J]. Blood,2007,110(5):1483-14910.
[58]YASUDA S, ATSUMI T, IEKO M, et al. Nicked beta 2-glycoprotein Ⅰ;a marker of cerebral infarct end a novel role in the negative feedLaek pathway of extrinsic fibrinolysis [J]. Blood,2004,103(10):3766-3772.
[59]Sttinkasserer T, Estaller C, Weiss EH, et al. Complete nucleotide and deduced amino acid sequence of human B2-glycoporotein 1. Biochem J 1991,277:387-391.
[60]Arrieux J, Roussel B, Jacob MC, et al. Measurement of antiphospholipol antibodies by ELISA using B2-ghycoprotein as an antigen J Immunol Methods 1991,143:223-229.
[61]Cabral AR, Alar con-stgovia D. Anti-beta 2 gly coprotein! Antibody testing in pationts with amtiphosphlipid syndome. Br J Rhoumato! 1997,36(11):1235.
[62]Tsutsumi A, Matsuura E, IchiKawa K, et al. IgA dass anti-beta-2-ghycoprotein in pationts with systemic lupus erythemtosus J khenmatol,1998,25(1):74-78.
[63]Kamboh, ML, Ferrell. RE and Sepehmia B. Genetic studies of human apolipoprotein. IV. Structural heterogeneity of apolipoprotein H. Am. J. Hum. Genet.1988,4(3):452-457.
[64]Haginara Y, Hong DP, Hoshino M, et al. Aggregation of beta2-glycoprotein I induced by sodium lauryl suffate and lysophos pholipids Buicgenstrt 2002 Jan 341(3): 1020-1026.
[65]Hunt J and Krilis S. The fifth domain of beta 2-glycoprotein I contains a phosphlipid binding site (Cys281-Cys288) and a region recognized by anticardiolipin antibodies. J Immunol.1994,152:653-659.
[66]Porter TF. Antiphospholipid antibodies and infertility. Clin Obstet Gynecol.2001,44: 29-35.
[67]Schwarzenbacher R, Zeth K, Diederichs K, et al. Crystal structure of human Β2-glycoprotein I:implications for phospholipid binding and the antiphospholipid syndrome. EMBO J.1999,18:6228-6239.
[68]Wurm H, Beubler E, Polz E. Studies on the possible function of beta2-glycoprotein 1: influence in the triglyceride metabolism in the rat Metabolism.1982,31(5):484-486.
[69]Brighton AT, Hogg PJ, Dai YP, et al. Beta-2-gylcoprotein I in thrombosis:evidence for a role as a natural anticoagulant. Br J Haemato,1996,93(1):185-194.
[70]Chonn A, Semple SC, Cullis PR. Beta-2-glycoprotein I is a major protein associated with very rapidly cheated? Liposomes in vivo, suggesting a significant role in the immune clearance of "non-self" particles. J Boil Chem.1995,270:25854-25859.
[71]Horhach D A, van Oort E, Donders K C, et al. Lupus amticoaguhnrt is the strongest risk factor for both venous amd arterial thrombosis in patients with svsteanic lupus ervtheanatosus. Comparison hetwexn different assays for the detection of mrtiphospholipid mrtibodies. Thromh Haemost,1996,76(6):916.
[72]Reher G, Arvieux J, Comby E, et al. Multicenter evaluation of nine commercial kits for the qumititation of amticnrdiolipin amtihodies. Thromh Haemost,1995,73(3):444.
[73]Me Neil HP, Smpson RJ, Chestenmm CN, et al. Antiphospholipid antibodies are directed agangainstnst a complex antigen that includes a lipid binding inhibitor of coagulation:β 2 Glycoprotein I (apolipoprotein H). Proc Natl Acad Sci LSA,1990, 87(11):4120.
[74]Tincan A, Spatola L, Balestrieri G. β 2GPI cofactor and ACA. Isr, J Med Sci,1995, 31(1):17.
[75]George J, Shoenfeld Y, Harats D. The involvement of beta 2-glycoprotein 1 (beta2-GP1) in human and murine atherosclerosis. J Autoimmun,1999,13:57-60.
[76]Finazzi G. The epidemiology of the antiphospholipid syndrome:who is at risk Curr Rheumatol Rep,2001,3:271-276.
[77]临床免疫学和免疫检验／王兰兰主编-北京-科学技术文献出版社,2004.10.
[78]P.G.DE GROOT, R.H.W.M. DERKSEN. Pathophysiology ofan-tiphospholipid antibodies [J]. The Netherlands journal of medicine,2004,62(8):267-272.
[79]JANINE J J. HULSTEIN, PETER J, et al. Beta2. Glycoprotein linhibits von Willebmnd factor-dependent platelet adhesion and aggregation[J]. Blood,2007,110(5):1483-1491.
[80]YASUDA S, ATSUMI T, IEKO M, et al. Nicked beta 2-glycoproteinI:a marker of 333 cerebral infarct and a novel role in the negativefeedba Sarmarco M, Soler C, Christides C.Prevalence and clinical significance of IgG isotope nati-beta2-glycoprotein 1 antibodies in anti-phospholipid syndrome[J]. J Lab Clin,1997,129(5):499-506.
[81]医学免疫学／金伯泉主编／第5版.人民卫生出版社,2008.6.
[82]Sakaguchi S.Naturally arising CD4+regulatory T cells for immunologic self-toleracnce and negative control of immune responses[J]. Annu Rev Immunol,2004,22:531.
[83]Shevach, EM. Regulatory T cells in autoimmunity[J]. annu Rev Immunol,2000,18:423.
[84]Picca CC, Caton AJ. The role of self-peptides in the development of CD4+CD25+ regulatory T cells[J]. Curr Opin Immunol,2005,17(4):131-136.
[85]付嘉,方艳秋,徐立,等.人β2糖蛋白1的重组表达及对抗磷脂抗体综合征患者血清诱导内皮细胞产生ICAM-1的影响[J].吉林大学学报(医学版),2006,32(4)：654-657.
[86]Angela T, Flavio A, Genesio B, et al. Minimal requirements for antiphospholipid antibodies ELISA proposed by the European Forum on antiphospholipid sntibodies[J]. Thrombosis Research,2004,114:553-558.
[87]Tolomeo T, Rico De Souza A, Roter E, et al. T cells demonstrate a Thl-biased response to native beta2-glycoprotein I in a murine model of anti-phospholipid antibody induction. Autoimmunity.2009,42(4):292-5.
[88]Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases[J]. Immunol,1995, 155(3):1151-1164.
[89]Banham AH. Cell-surface IL-7 receptor expression facilitates the purification of Foxp3+ regulatory T cells [J]. Trends Imm unol,2006,27(12):541-544.
[90]Corthay A. How do regulatory T cells work Scand J Immunol.2009 Oct, 70(4):326-36.
[91]孟荔,欧阳建.CD4+CD25+调节性T细胞与自身免疫病.中国组织工程研究与临床康复,2007,11(33)：6676-6680.
[92]D. M. Waid, G. M. Vaitait is, N. D. Pennock, et al. Disruption of the homeostatic balance between autoaggressive (CD4+CD40+) and regulatory (CD4+CD25+FoxP3+) T cells promotes diabetes. J. Leukoc. Biol. August 1,2008,84(2):431-439.
[93]Kuhn A, Beissert S, Krammer PH. CD4+CD25+regulatory T cells in human lupus erythematosus. Arch Dermatol Res.2009 Jan,301(1):71-81.
[94]Moldigilin Wan S, Xia C, Morel L. IL-6 p roduced by dendritic cells from lupus-prone miceinhibits CD4+CD25+T cell regulatory functions[J]. J Imm unol,2007,178(1):271-279.
[95]Valencia X, Stephens G, Goldbach Mansky R, et al. TNF down-modulates the function of human CD4+CD25hi T regulatory cells[J]. Blood,2006,108(1):253-261.
[96]Pillai V, Ortega SB, Wang CK, et al. Transient regulatory T cells:A state attained By all activeated human T cells[J]. Clin Immumol,2007,123:18-29.
[97]Sakaguchi S. Naturally arisiy CD4+regulatory T cells for immunologic self-tolerance and negative control of immune responses [J]. Annu Rev Immunol,2004,22:531-562.
[98]Construction and characterization of a novel DNA vaccine that is potent antigen-specific tolerizing therapy for experimental arthritis by increasing CD4+CD25+Treg cells and inducing Thl to Th2 shift in both cells and cytokine(song X, Liang F, Liu N, Luo Y, Xue H, Yuan F, Tan L, Sun Y, Xi C, Xi Y.Vaccine.2009 Jan 29,27(5):690-700. Epub 2008 Dec 16.)
[99]Zhang B, Sun C, Qu Y, et al. Deficiency of mouse CD4+CD25+Foxp3+regulatory T cells in xenogeneic pig thymus-grafted nude mice suffering from autoimmune diseases. Cell Mol Immunol.2008 Oct,5(5):325-32.
[100]Hall HT, Sjolin H, Brauner H, et al. Increased diabetes development and decreased function of CD4+CD25+Treg in the absence of a functional DAP 12 adaptor proteinEur J Immunol.2008 Nov,38(11):3191-9.
[101]Identification of correlations between numbers of CD4+CD25+Treg cells, levels of sera anti-AChR antibodies and transfer growth factor-beta in patients with myasthenia gravis (Wang HB, Zhang JB, Chui LY. Zhonghua Yi Xue Za Zhi.2008 Apr 15,88(15):1036-40. Chinese)
[102]Dysfunctional CD4+, CD25+regulatory T cells in untreated active systemic lupus erythematosus secondary to interferon-alpha-producing antigen-presenting cells (Yan B, Ye S, Chen G, Kuang M, Shen N, Chen S. Arthritis Rheum.2008 Mar,58(3):801-12.
[103]CD4+CD25+regulatory T cells in health and disease (Liu H, Leung BP.Clin Exp Pharmacol Physiol.2006 May-Jun; 33(5-6):519-24. Review)
[104]Paralysis of CD4+CD25+regulatory T cell response in chronic autoimmune encephalomyelitis (Matsumoto Y, Sakuma H, Kohyama K, Park IK. J Neuroimmunol. 2007 Jul; 187(1-2):44-54. Epub 2007 May 17.)