融合蛋白CTLA4Ig对再生障碍性贫血小鼠骨髓T细胞功能的作用研究
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摘要
第一部分
再生障碍性贫血小鼠骨髓T细胞表面CD28、CTLA4水平检测及其意义
目的:建立再生障碍性贫血小鼠模型。体外观察再障小鼠骨髓T细胞表面CD28、CTLA4的表达变化及其意义。
方法建立免疫介导再障小鼠模型,分离再障小鼠和正常组小鼠骨髓单个核细胞(BMMNC),体外利用植物血凝素(PHA,终浓度为15μg/ml)特异性激活T细胞,用双色免疫荧光标记流式细胞术检测正常小鼠和再障小鼠于PHA刺激前后骨髓T细胞表面CD28和CTLA4的表达水平。
结果PHA刺激培养48小时后,正常小鼠和再障小鼠骨髓T细胞表面CD28和CTLA4的表达较刺激前均明显升高(P<0.0 1);而且再障小鼠PHA刺激培养前后T细胞CD28的表达均显著高于同期正常小鼠(P<0.0 1),但CTLA4的表达与同期正常小鼠相比虽略有升高,但差异无显著性(P>0.05)。
结论再障小鼠骨髓T细胞激活及其激活潜能增强,免疫分子CD28和CTLA4在骨髓中的异常表达可能参与了再障T细胞免疫功能紊乱,或是紊乱的表现之一。
第二部分
再生障碍性贫血小鼠骨髓淋巴细胞Th1/Th2细胞亚群漂移与转录因子T-bet和GATA-3的调控作用
目的:探讨再障小鼠骨髓淋巴细胞的Th1/Th2细胞亚群的漂移与转录因子T-bet和GATA-3的调控作用。
方法:分离正常小鼠和再障小鼠的骨髓淋巴细胞,在终浓度为15μg/ml植物血凝素(PHA)条件下培养,用双抗体夹心ELISA方法检测小鼠骨髓淋巴细胞培养上清中Th1类细胞因子IFN-γ和Th2类细胞因子IL-4的水平;用RT-PCR技术检测小鼠骨髓淋巴细胞IFN-γ、IL-4以及转录因子T-bet和GATA-3 mRNA表达强度;Western Blot方法检测T-bet和GATA-3的蛋白表达水平。
结果:①再障小鼠骨髓淋巴细胞分泌Th1/Th2类细胞因子INF-γ和IL-4浓度及其在淋巴细胞中mRNA的表达水平均显著高于正常组(P<0.0 1),而IL-4基因和蛋白水平明显低于正常组(P<0.0 1);②再障小鼠骨髓淋巴细胞转录因子T-bet基因和蛋白水平显著高于正常组(P<0.0 1),而GATA-3基因和蛋白水平明显低于正常组(P<0.0 1);相关性分析显示,再障小鼠IFN-γ浓度与T-bet蛋白水平呈正相关,与GATA-3无相关性,而L-4浓度与GATA-3呈正相关,与T-bet呈负相关。
结论:再障小鼠骨髓出现T细胞向Th1亚群漂移现象,导致机体Th1细胞的优势应答状态,从而诱导细胞毒性T细胞对造血干细胞的细胞毒作用,而T-bet表达增强与GATA-3低表达可能是再障小鼠骨髓Th1优势分化的重要原因。
第三部分
融合蛋白CTLA4Ig对再生障碍性贫血小鼠骨髓T细胞功能的作用研究
目的:观察CTLA4Ig对再生障碍性贫血小鼠骨髓T细胞增殖、Th1和Th2类细胞因子表达水平以及T细胞对靶细胞的细胞毒活性的影响。
方法:利用同种异体来源的树突状细胞(DC)作为抗原和抗原提呈细胞,在终浓度为25μg/ml的丝裂霉素C处理后作为刺激细胞,尼龙毛方法分离正常小鼠和再障小鼠骨髓的T细胞作为反应细胞,进行单向混合淋巴细胞培养(MLR),同时加入终浓度为10μg/ml的CTLA4Ig共培养,MTT比色法检测CTLA4Ig对T细胞增殖的影响,ELISA方法检测MLR培养上清Th1和Th2类细胞因子水平变化;体外诱导CTL,乳酸脱氢酶试验检测CTLA4Ig对再障骨髓CTL细胞毒活性的影响。
结果:CTLA4Ig可以在体外明显抑制再障小鼠过度的T细胞增殖,并使其降至正常水平;可以下调Th1类细胞因子,上调Th2类细胞因子,诱导再障小鼠骨髓Th1/Th2失衡向Th2的转换,使再障Th1异常优势应答状态恢复正常;抑制再障小鼠CTL细胞的杀伤活性。
结论:CTLA4Ig能够在体外抑制再障小鼠骨髓T细胞的增殖、诱导Th1/Th2的转换,恢复再障Th1异常优势应答状态,抑制骨髓CTL的杀伤活性,从而恢复骨髓造血。
Part Ⅰ
Expressions of CD28 and CTLA4 on T cells from bone morrow in aplastic anemia mice.
Objective: To investigate the expressions and significance of CD28 and CTLA4 on T cells in bone marrow of aplastic anemia (AA) mice,
Methods: In vitro bone marrow mononuclear cells (BMMNCs) were activated through being incubated with PHA (15μg/ml). The CD28 and CTLA4 expressions on T cells incubated with or without PHA were analyzed by two color flow cytometry. The expressions of CD28 and CTLA4 significantly increased after PHA stimulation. In the AA mice, the expressions of CD28 with or without PHA stimulation were both higher than that in the normal mice (P<0.01 and P<0.01), but the expressions of CTLA4 with or without PHA stimulation had both no significant difference compared with that in the normal mice (P>0.05 and P>0.05). In the AA mice, there were more activation and activated potential of T cell than the normal, and the abnormal expressions of CD28 and CTLA4 maybe participate in immunological disorder mediated by T cell. Part Ⅱ
T cell specific transcription factors T-bet and GATA-3 contribute to shifted development of T help cell toward type I from bone marrow in aplastic anemia (AA) mice
Objective: To investigate the T helper cell predominant differentiation in aplastic anemia (AA) mice and to explore the modulation of T cell specific transcription factors T-bet and GATA-3.
Methods: Established model of AA mice. Lymphocytes were isolated from bone marrow incubated with phytohemagglutinin (PHA)(15μg/ml) in vitro. Cytokines IL-2, IFN-γ, IL-4 and IL-10 concentration in bone marrow were detected by ELISA. The gene expression levels of transcription factor T-bet/GATA-3 were assayed by RT-PCR.
Results: In AA mice, the levels of IL-2 and IFN-γ were markedly increased but IL-4 and IL-10 were markedly decreased than normal mice. And the gene and protein expressions of T-bet in AA mice were much higher than normal, but GATA-3 were lower than normal.
Conclusion: In AA mice, there is a shifted development of Th toward to Th1, which maybe involved in induction of T-cells cytotoxic to hematopoietic stem cell. Furthermore, type I shift of Th cause by the level of T-bet was significantly over expressed and GATA-3 decreased. Part Ⅲ
Effect of CTLA4Ig on T cells which from bone marrow in aplastic anemia (AA) mice in vitro
Objective: To study the effect of CTLA4Ig on T cells which from bone marrow in aplastic anemia mice in vitro.
Methods: T cells from bone marrow of normal and AA mice (as reaction cells) with same amount of mitocin-C treated DC (as stimulation cells) were co-cultured for 5 days in the presence CTLA4Ig. MTT colorimetry was used to detect allogeneic T cells proliferation. Using ELISA assay to detect the level of allogeneic response T cells cytokine: IFN- γ and IL-4. Co-cultured the above allogeneic response T cells (as stimulation cells) with DC (as reaction cells), using LDH Cytotoxicity Assay Kit detect the cytotoxic activity of CTL.
Result: In aplastic anemia the proliferation response of T cells induced by DC and in presence CTLA4Ig was significantly lower than which without CTLA4Ig, the level of INF- γ was markedly decreased and the level of IL-4 markedly increased by CTLA4Ig, the cytotoxic activity was markedly decreased by CTLA4Ig in vitro.
Conclusion: In aplastic anemia, CTLA4Ig could inhibit T cells proliferation, down-regulate the Th1 cytokines and up-regulate the Th2 cytokines, and inhibit the cytotoxic activity in vitro, so that induced T cell anergy by blocking B7/CD28pathway.
再生障碍性贫血小鼠骨髓T细胞表面CD28、CTLA4水平检测及其意义
目的:建立再生障碍性贫血小鼠模型。体外观察再障小鼠骨髓T细胞表面CD28、CTLA4的表达变化及其意义。
方法建立免疫介导再障小鼠模型,分离再障小鼠和正常组小鼠骨髓单个核细胞(BMMNC),体外利用植物血凝素(PHA,终浓度为15μg/ml)特异性激活T细胞,用双色免疫荧光标记流式细胞术检测正常小鼠和再障小鼠于PHA刺激前后骨髓T细胞表面CD28和CTLA4的表达水平。
结果PHA刺激培养48小时后,正常小鼠和再障小鼠骨髓T细胞表面CD28和CTLA4的表达较刺激前均明显升高(P<0.0 1);而且再障小鼠PHA刺激培养前后T细胞CD28的表达均显著高于同期正常小鼠(P<0.0 1),但CTLA4的表达与同期正常小鼠相比虽略有升高,但差异无显著性(P>0.05)。
结论再障小鼠骨髓T细胞激活及其激活潜能增强,免疫分子CD28和CTLA4在骨髓中的异常表达可能参与了再障T细胞免疫功能紊乱,或是紊乱的表现之一。
第二部分
再生障碍性贫血小鼠骨髓淋巴细胞Th1/Th2细胞亚群漂移与转录因子T-bet和GATA-3的调控作用
目的:探讨再障小鼠骨髓淋巴细胞的Th1/Th2细胞亚群的漂移与转录因子T-bet和GATA-3的调控作用。
方法:分离正常小鼠和再障小鼠的骨髓淋巴细胞,在终浓度为15μg/ml植物血凝素(PHA)条件下培养,用双抗体夹心ELISA方法检测小鼠骨髓淋巴细胞培养上清中Th1类细胞因子IFN-γ和Th2类细胞因子IL-4的水平;用RT-PCR技术检测小鼠骨髓淋巴细胞IFN-γ、IL-4以及转录因子T-bet和GATA-3 mRNA表达强度;Western Blot方法检测T-bet和GATA-3的蛋白表达水平。
结果:①再障小鼠骨髓淋巴细胞分泌Th1/Th2类细胞因子INF-γ和IL-4浓度及其在淋巴细胞中mRNA的表达水平均显著高于正常组(P<0.0 1),而IL-4基因和蛋白水平明显低于正常组(P<0.0 1);②再障小鼠骨髓淋巴细胞转录因子T-bet基因和蛋白水平显著高于正常组(P<0.0 1),而GATA-3基因和蛋白水平明显低于正常组(P<0.0 1);相关性分析显示,再障小鼠IFN-γ浓度与T-bet蛋白水平呈正相关,与GATA-3无相关性,而L-4浓度与GATA-3呈正相关,与T-bet呈负相关。
结论:再障小鼠骨髓出现T细胞向Th1亚群漂移现象,导致机体Th1细胞的优势应答状态,从而诱导细胞毒性T细胞对造血干细胞的细胞毒作用,而T-bet表达增强与GATA-3低表达可能是再障小鼠骨髓Th1优势分化的重要原因。
第三部分
融合蛋白CTLA4Ig对再生障碍性贫血小鼠骨髓T细胞功能的作用研究
目的:观察CTLA4Ig对再生障碍性贫血小鼠骨髓T细胞增殖、Th1和Th2类细胞因子表达水平以及T细胞对靶细胞的细胞毒活性的影响。
方法:利用同种异体来源的树突状细胞(DC)作为抗原和抗原提呈细胞,在终浓度为25μg/ml的丝裂霉素C处理后作为刺激细胞,尼龙毛方法分离正常小鼠和再障小鼠骨髓的T细胞作为反应细胞,进行单向混合淋巴细胞培养(MLR),同时加入终浓度为10μg/ml的CTLA4Ig共培养,MTT比色法检测CTLA4Ig对T细胞增殖的影响,ELISA方法检测MLR培养上清Th1和Th2类细胞因子水平变化;体外诱导CTL,乳酸脱氢酶试验检测CTLA4Ig对再障骨髓CTL细胞毒活性的影响。
结果:CTLA4Ig可以在体外明显抑制再障小鼠过度的T细胞增殖,并使其降至正常水平;可以下调Th1类细胞因子,上调Th2类细胞因子,诱导再障小鼠骨髓Th1/Th2失衡向Th2的转换,使再障Th1异常优势应答状态恢复正常;抑制再障小鼠CTL细胞的杀伤活性。
结论:CTLA4Ig能够在体外抑制再障小鼠骨髓T细胞的增殖、诱导Th1/Th2的转换,恢复再障Th1异常优势应答状态,抑制骨髓CTL的杀伤活性,从而恢复骨髓造血。
Part Ⅰ
Expressions of CD28 and CTLA4 on T cells from bone morrow in aplastic anemia mice.
Objective: To investigate the expressions and significance of CD28 and CTLA4 on T cells in bone marrow of aplastic anemia (AA) mice,
Methods: In vitro bone marrow mononuclear cells (BMMNCs) were activated through being incubated with PHA (15μg/ml). The CD28 and CTLA4 expressions on T cells incubated with or without PHA were analyzed by two color flow cytometry. The expressions of CD28 and CTLA4 significantly increased after PHA stimulation. In the AA mice, the expressions of CD28 with or without PHA stimulation were both higher than that in the normal mice (P<0.01 and P<0.01), but the expressions of CTLA4 with or without PHA stimulation had both no significant difference compared with that in the normal mice (P>0.05 and P>0.05). In the AA mice, there were more activation and activated potential of T cell than the normal, and the abnormal expressions of CD28 and CTLA4 maybe participate in immunological disorder mediated by T cell. Part Ⅱ
T cell specific transcription factors T-bet and GATA-3 contribute to shifted development of T help cell toward type I from bone marrow in aplastic anemia (AA) mice
Objective: To investigate the T helper cell predominant differentiation in aplastic anemia (AA) mice and to explore the modulation of T cell specific transcription factors T-bet and GATA-3.
Methods: Established model of AA mice. Lymphocytes were isolated from bone marrow incubated with phytohemagglutinin (PHA)(15μg/ml) in vitro. Cytokines IL-2, IFN-γ, IL-4 and IL-10 concentration in bone marrow were detected by ELISA. The gene expression levels of transcription factor T-bet/GATA-3 were assayed by RT-PCR.
Results: In AA mice, the levels of IL-2 and IFN-γ were markedly increased but IL-4 and IL-10 were markedly decreased than normal mice. And the gene and protein expressions of T-bet in AA mice were much higher than normal, but GATA-3 were lower than normal.
Conclusion: In AA mice, there is a shifted development of Th toward to Th1, which maybe involved in induction of T-cells cytotoxic to hematopoietic stem cell. Furthermore, type I shift of Th cause by the level of T-bet was significantly over expressed and GATA-3 decreased. Part Ⅲ
Effect of CTLA4Ig on T cells which from bone marrow in aplastic anemia (AA) mice in vitro
Objective: To study the effect of CTLA4Ig on T cells which from bone marrow in aplastic anemia mice in vitro.
Methods: T cells from bone marrow of normal and AA mice (as reaction cells) with same amount of mitocin-C treated DC (as stimulation cells) were co-cultured for 5 days in the presence CTLA4Ig. MTT colorimetry was used to detect allogeneic T cells proliferation. Using ELISA assay to detect the level of allogeneic response T cells cytokine: IFN- γ and IL-4. Co-cultured the above allogeneic response T cells (as stimulation cells) with DC (as reaction cells), using LDH Cytotoxicity Assay Kit detect the cytotoxic activity of CTL.
Result: In aplastic anemia the proliferation response of T cells induced by DC and in presence CTLA4Ig was significantly lower than which without CTLA4Ig, the level of INF- γ was markedly decreased and the level of IL-4 markedly increased by CTLA4Ig, the cytotoxic activity was markedly decreased by CTLA4Ig in vitro.
Conclusion: In aplastic anemia, CTLA4Ig could inhibit T cells proliferation, down-regulate the Th1 cytokines and up-regulate the Th2 cytokines, and inhibit the cytotoxic activity in vitro, so that induced T cell anergy by blocking B7/CD28pathway.
引文
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13 Maciejewski JP, Selleri C, Sato T, et al. Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anemia. Br J Haematol.1995; 91:245-252.
14 Romagnami S. Human Th1 and Th2 subsets: doubt no more. Immnnol Today, 1991, 12: 256-257.
15 Romagnami S. T cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol, 2000,85:9-18.
16 Haggqvist B, Hultman P. Effects of deviating the Th2 response in murine mercury induced autoimmunity towards a Thl response. Clin Exp Immuol, 2003, 134:202-209.
17 Dominguez-Garcia MV, Rodriguez-Moyado H. Cellular and biochemical mechanisms involved in physiopathgenesis of auto-immune thrombocytopenic purpura. Gac Med Mex, 2002, 138: 461-472.
18 Szabo SJ, Kim ST, Costa GL, et al. A novel transcription factor, T-bet, directs Thl linage commitment. Cell, 2000, 100: 655-669.
19 邢同京，章廉，主编．TH 类细胞极化群体的基础与临床．北京：军事医学科学出版社，2002，35-41．
20 Dorfman DM, Vanden-Elzen P, Weng AP, et al. Differential expression of T-bet, a T-box transcription factor required for T_H1 T-cell development, in peripheral T-cell lymphomas. Am J Clin Pathol, 2003, 120: 866-873.
21 Zheng WP, Flavell RA. The Transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CO_4~+ T cells. Cell, 1997, 89: 587-599.
22 Kaito K, Otsubo H, Usui N, et al. Th1/Th2 lymphocyte balance in patients with aplastic anemia. Rinsho Byori. 2004 ,52(7):569-73.
23 Giannakoulas NC, Karakantza M, Theodorou GL, et al. Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients. Br J Haematol. 2004, 124(1): 97-105.
1 Nakao S. Immune mechanism of aplastic anrmia. Int J Hematol 1997, 66:127-134.
2 Jaroslaw P, Maciejewski, Antomnio R, et al. Immune Pathophysiology of Aplastic Anemia.Inter J of Hematol, 2002, 76207-214.
3 Immunization with dendritic cells pulsed with tarnor extract increases survival of mice bearing intracranial gliomas. Ni HT, Spellman SR, Jean WC, et al. J Neurooncol. 2001, 51(1):1-9.
4 Cloned Dendritic Cells Can Present Exogenous Antigens on Both MHC Classs Ⅰ and Class Ⅱ Molecules. Zhenhai S, Glen R, Glenn D, et al. J Immunol. 1997, 158:2723-2730.
5 薛庆善主编．体外培养的原理与技术．北京：科学出版社，2001，201-201．
6 Young NS, Maciejewski J. The pathophysiology of acquired aplastic anemia. N Engl J Med. 1997, 336:1365—1372.
7 Vfolk A, Simon-Stoos K, Nami I, Concannon J, Mawe J, T anawattanacha-roen P, et al. A mouse model of immune-mediated aplastic anemia. Blood. 1998, 10(Suppl 1): 158a-159a.
8 Hoon Kook, Antonio M, Risitano WZ, et al. Changes in T-cell receptor VB repertoire in aplastic anemia: effects of different immunosuppressive regimens. Blood. 2002, 99:3668-3675.
9 Harding FA, McArthur JR, Gross JA, et al. CD28 mediated signaling costimulates murine T cells and prevents induction of anergy in T cell clones. Nature,1992;356:607-609.
10 Bugeon L, Dallman MJ. Costimulation of T cells. Am J Respir Crit Care Med, 2000, 162(4 Pt 2):S164-172.
11 Karandikar NJ, Vanderlugt CL, Walunas TL, et al. CRLA4:a negative regulator of autoimmune disease. J Exp Med, 1996, 184:783-792.
12 Balow JE, Boumpas DT, Austin HA, et al. New prospects for treatment of lupus nephritis. Semin Nephrol, 2000, 20: 32- 39.
13 Reynolds J, Tam FW, Chandraker A, et al. CD28-B7 blockade prevents the development of experimental auto-immune glomerulonephritis. J Clin Invest. 2000, 105: 643-651.
14 Perrin PJ, June CH, Maldonado JH, et al. Blockade of CD28 durinn in vitro activation of encephalitogenic T cells or after disease nset ameliorates experimental auto- immune encephalomyelitis. J Immunol. 1999, 163: 1704-1710.
15 Sato J, Asakura K, Murakami M, et al. Topical CTLA4- Ig suppresses ongoing mucosal immune response in pre-sensitized murine model of allergic rhinitis. Int Arch Allerny Immunol. 1999, 119:197-204.
16 Kaito K, Otsubo H, Usui N, et al. Th1/Th2 lymphocyte balance in patients with aplastic anemia. Rinsho Byori. 2004,52: 569-73.
17 Giannakoulas NC, Karakantza M, Theodorou GL, et al. Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients. Br J Haematol. 2004,124: 97-105.
18 Linslev PS, Wallace PM, Joneson J, et al. Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule. Science, 1992, 257(5071):792- 795.
1. Nakao S. Immune mechanism of aplastic anrmia.Int J Hematol 1997, 66:127-134.
2. Jaroslaw P, Maciejewski, Antomnio R, et al. Immune Pathophysiology of Aplastic Anemia.Inter J of Hematol, 2002, 76207-214.
3. Chiu KM, Knospe WH. Immunological mediated aplastic anemia mice: effects of varying the source and composition of donor cell. Exp Hematol, 1987, 15:269-275.
4. Melenhorst JJ, van Krieken JHJM, Dreef E, et al. T cell selectively infiltration bone marrow areas with residual haemopoiesis of patients with acquired aplastic anaemia. Br J Haematol, 1997, 99:527-519.
5. Viale M, Merli A, Bacigalupo A.Analysis at the clonal level of T-cell phenotype and function in sever aplastic anemia patients. Blood, 1991,78:1268-1274.
6. Macieje wski JP, Hibbs JR, Anderson S, et al. Blood marrow and peripheral blood lymphocyte phenotype in patients with bone marrow failure. Exp Hematol 1994, 22:1102-1110.
7. Hoon K, Weihua Z, Chen G, et al. Increased cytotoxic T cell with effector phenotype in aplastic anemia and myelodysplasia. Exp Hematol.2001, 29:1270-1277.
8. Hsu HC, Tsai WH, Chen LY, et al. Overproduction of inhibitory hematopoietic cytokines by lipoplysaccharide-activatde peripheral blood mononuclear cell in patients with aplastic anemia. Ann Hematol, 1995, 71:281-286.
9. Nistico A, Young NS, Gamma-interferon gene expression in the bone marrow of patients with aplastic anemia. Ann Intern Med, 1994, 120:463-469.
10.Nakao S, Yamaguchi M, Shiobara S, et al. Interferon-r gene expression in unstimulated bone marrow mononuclear cells predicts a good response to cycloporine therapy in aplastic anemia. Blood, 1992, 79:2532-2535.
11.Hiaoyuki T and Hiroshi Y. Type I and Type II T-cell Profiles in Aplastic Anemia and Refractory Anemia. American J of Hematol, 2000, 64:271-274.
12. Manz CY, Dietrich PY, Schnuriger V, et al. T-cellreceptor beta chain variability in bone marrow andperipheral blood in severe acquired aplastic anemia. Blood Cell S Mol Dis, 1997;23:110-121.
13. Zeng W, Nakao S, Takamatsu H, et al.Characterization of T-cellrepertoire of bonemarrow in immune mediated aplastic anemia revidence for the involve ment of antigen-drive T-cellresponse in cyclosporine-dependentaplastic anemia.Blood, 1999,93(9)::3008-3016.
14. Nimer SD, Ireland P, Meshikinpour AP, et al. An increased HLA-DR2frequency is seen in aplastic anemia.Blood,1994,84:923-92
15. Nakao S, Takamatsu H, Chuhjo T, et al. Identification of a specific HLA class II haplotype strongly associated with susceptibility to cyclosporine-dependent aplastic anemia. Blood, 1994, 84:4257-4261.
16. Nakao S, Takami A, Sugimori N, et al .Response to immunosppressive therpy and an HLA-DRB1++1501 does not predict reponse to antithymocyte globulin. Br J Haemetol, 1996, 92:155-158.
17. Genestier L, Fournel S, Flacher M, et al. Induction of Fas-mediated apoptosis of activated lyphocytes by polyclonal antithy mocyte globulins. Blood, 1998, 91:2360-2368.
1 Linsley PS, Wallace PM, Johnson J, et al. Immunosupression in vivo by a soluble form of the CTLA4 T cell activation molecule. Science. 1992,257:792-795.
2 Janeway CA, Bottornley K. Signals and signs for lymphocyte responses. Cell, 1994, 76:275-280.
3 范祖森．共刺激信号与免疫耐受．国外医学·免疫学分册．1997，20：244-247．
4 Wu Y, Guo Y, Liu Y, et al. A major costimulatory molecule on antigen-pressenting cells, CTLA4 ligand a isdistinct from B7. J Exp Med. 1993, 178:1789-1796.
5 Hathcock KS, Laszlo G, Dickler HB, et al. Indentifiction of analternative CTLA-4 ligand costimulatory for cell activation. Science.1993, 262:905-909.
6 Linsley PS, Brady W, Grosmarie L, et al. Binding of the B cellactivation antigen B7 to CD28 costimulates T cell proliferation and interleakin 2 mRNA accumulation. J Exp Med. 1991, 173:721-730.
7 TurkaL A, Linsley PS, Lin H, et al. T-cell activation by the CD28 ligand B7 is required for cardiac allograft rejection in vivo. Proc NatlAcad Sci USA. 1992,89:11102-11108.
8 Lenschow DJ, Zeng Y, Thistlethwaite JR, et al. Long-term survival of xenogeneic pancreaticislet grafts induced by CTLA4Ig. Science. 1992, 257:789-796.
9 Bierer BE, Hollander G, Fruman D, et al. Cyclosporin A and FK506: molecular mechanism of immunosuppression and probes for transplantationbiology. Curr Opin Immunol.1993, 5:763-771.
10 Lenschow DJ, Zeng Y, Thistlethwaite JR, et al. Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4Ig. Scence.1992, 257:789-792.
11 Hirakawa E, Yasunami Y, Nakano M, et al. Amelioration of hyperglycemia in streptozotocin-induced diabetic mice with fetal pancreatic allografts: prevention of rejection by donor specific transfusion in conjunction with CTLA4Ig. Pancreas. 2004, 28:146-152.
12 Reddy B, Gupta S, Chuzhin Y, et al. The effect of CD28/B7 blockade on alloreactive T and B cells after liver cell transplantation. Transplantation. 2001, 71:801-11.
13 Kimura F, Gotoh M, Tanaka T, et al. Locally expressed CTLA4Ig in a pancreatic beta-cell line suppresses accelerated graft rejection response induced by donor-specific transfusion. Diabetologia. 2000 .45:831-840.
14 Guinan EC, Boussiotis VA, Neuberg D, et al. Transplantation of anergic histoincompatible bone marrow allografts. N Engl J Med. 1999, 340:1704-1714.
15 Romagnami S. Human Th1 and Th2 subsets: doubt no more. Immnnol Today,.1991, 12: 256-257.
16 Romagnami S. T cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol, 2000, 85:9-18.
17 Haggqvist B, Hultman P. Effects of deviating the Th2 response in murine mercury induced autoimmunity towards a Th1 response. Clin Exp Immuol, 2003, 134:202-209.
18 Dominguez-Garcia MV, Rodriguez-Moyado H. Cellular and biochemical mechanisms involved in physiopathgenesis of auto-immune thrombocytopenic purpura. Gac Med Mex, 2002, 138: 461-472.
19 Kita Y, Li XK, Ohba M, et al. Prolonged cardiac allograft survival in rats systemically injected adenoviral vector scontaining CTLA4Ig-gene. Transplantation. 1999,68:758-766.
20 McIntosh KR, Linsley PS, Bacha PA, et al. Immunotherapy of experimental autoimmune myasthenia gravis: selective effects of CTLA4Ig and synergistic combination with an IL2-diphtheria toxin fusion protein. J Neuroimmunol. 1998 Jul 1;87(1-2):136-46.。
21 BalowJ E, Boumpas DT, Austin HA. New prospects for treatment of lupusnephritis. Semin Nephrol.2000, 20:32-39.
22 Perrin PJ, June CH, Maldonado JH, et al. Blockade of CD28 during in vitro activation of encephalitogenic T cells or after disease onset ameliorates experimental auto-immune encephalomyelitis.J Immunol.1999,163:1704-1710.
23 Reynolds J, Tam FW, Chandraker A, et al. CD28-B7 blockade prevents the development of experimental auto-immune glomerulonephritis. J Clin Invest. 2000,105:643-651.
24 Sato J, Asakura K, Murakami M, et al. Topical CTLA4Ig suppresses ongoing mucosal immune response in pre-sensitized murine model of allergic rhinitis. Int Arch Allergy Immunol. 1999,119:197-204.
25 Abrams JR, Lebwohl MG, Guzzo CA, et al. CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris.J Clin Invest. 1999,103:1243-52.
26 Abrams JR, Kelley SL, Jegasothy BV, et al. Blockade of T lymphocyte costimulation with cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (CTLA4Ig) reverses the cellular pathology of psoriatic plaques, including the activation of keratinocytes, dendritic cells, and endothelial cells.J Exp Med. 2000 ,192:681-694.
27 邱霖．百时美施贵宝公司发布类风湿关节炎治疗新药 CTLA4Ig 的临床结果．上海医药，2002，23：293．
28 Mohamed H, Sayegh, Finally. CTLA4-Ig graduates to the clinic. J Clin Invest. 1999,103:1223-1225.
29 Gebhardt BM, Hodkin M, Varnell ED, et al. Protection of corneal allografts by CTLA4-Ig. Cornea. 1999, 18(3):314-20.
2 Wolk A, Simon-Stoos K, Nami I, et al. A mouse model of immune-mediated aplastic anemia. Blood.1998; 10(Suppl l):158a-159a.
3 Hoon Kook; Antonio M. Risitano, Weihua Zeng, et al. Changes in T-cell receptor VB repertoire in aplastic anemia: effects of different immunosuppressive regimens. Blood.2002, 99:3668-3675.
4 Nakao S. Immune mechanism of aplastic anrmia.Int J Hematol 1997, 66:127-134.
5 Jaroslaw P, Maciejewski, Antomnio R, et al. Immune Pathophysiology of Aplastic Anemia.Inter J of Hematol, 2002, 76207-214.
6 Harding FA, McArthur JR, Gross JA, et al. CD28 mediated signaling costimulates murine T cells and prevents induction of anergy in T cell clones. Nature, 1992;356:607-609.
7 Bugeon L, Dallman MJ. Costimulation of T cells. Am J Respir Crit Care Med, 2000, 162(4 Pt 2):S164-172.
8 Karandikar NJ, Vanderlugt CL, Walunas TL, et al. CRLA4:a negative regulator of autoimmune disease. J Exp Med, 1996, 184:783-792.
9 Shiraishi T, Yasunami Y, Takehara M, et al. Prevention of acute lung allograft rejection in rat by CTLA4Ig. Am J Transplant. 2002,2:223.
10 Hayashi S, Guang LinM, Yokoyama I, et al. Adenovirus mediated genetransfer of CTLA4Ig gene result in prolonged survival of heart allograft. Transpl Int. 2000, 1:S329.
11 Ajiki T, Takahashi M, Hakamata Y,Difficulty of achieving long-term graft survival of MHC-disparate composite graft using CTLA4IG.Transplantation. 2003, 76:438; 438-9.
12 Watanabe T.Adenovirus-mediated CTLA4Ig gene therapy in cardiac xenotransplantationHokkaido Igaku Zasshi. 2004,79:47-53.
13 Balow JE, Boumpas DT, Austin HA, et al. New prospects for treatment of lupus nephritis. Semin Nephrol, 2000, 20: 32- 39.
14 Reynolds J, Tarn FW, Chandraker A, et al. CD28-B7 blockade prevents the development of experimental auto-immune glomerulonephritis. J Clin Invest. 2000, 105: 643-651.
15 Perrin PJ, June CH, Maldonado JH, et al. Blockade of CD28 durinn in vitro activation of encephalitogenic T cells or after disease onset ameliorates experimental auto- immune encephalomyelitis. J Immunol. 1999, 163: 1704-1710.
16 Sato J, Asakura K, Murakami M, et al. Topical CTLA4- Ig suppresses ongoing mucosal immune response in pre-sensitized murine model of allergic rhinitis. Int Arch Allerny Immunol. 1999, 119:197-204.
1 Young NS, Maciejewski J. The pathophysiology of acquired aplastic anemia. N Engl J Med. 1997, 336: 1365-1372.
2 Vfolk A, Simon-Stoos K, Nami I, Concannon J, Mawe J, T anawattanacha-roen P, et al. A mouse model of immune-mediated aplastic anemia. Blood. 1998, 10(Suppl 1): 158a-159a.
3 Hoon Kook, Antonio M, Risitano WZ, et al. Changes in T-cell receptor VB repertoire in aplastic anemia: effects of different immunosuppressive regimens. Blood. 2002, 99:3668-3675.
4 Harding FA, McArthur JR, Gross JA, et al. CD28 mediated signaling costimulates murine T cells and prevents induction of anergy in T cell clones. Nature. 1992, 356:607-609.
5 Bugeon L, Dallman MJ. Costimulation of T cells. Am J Respir Crit Care Med, 2000, 162(4 Pt 2):S164-172.
6 姚军，李树浓．淋巴细胞与再生障碍性贫血的实验研究．中华血液学杂志，1991，12：229-231．
7 Janeway CA, Bottornley K. Signals and signs for lymphocyte responses. Cell, 1994, 76:275-280.
8 范祖森．共刺激信号与免疫耐受．国外医学·免疫学分册．1997，20：244-247．
9 Nitta K, Horita S, Ogawa S, et al. Resistance of CD28-deficient mice to autologous phase of anti-glomerular basement membrane glomerulonephritis. Clin Exp Nephrol. 2003, 7(2):104-12.
10 Wu Y, Guo Y, Liu Y, et al. A major costimulatory molecule on antigen-pressenting cells, CTLA4 ligand a isdistinct from B7. J Exp Med. 1993, 178:1789-1796.
11 Hathcock KS, Laszlo G, Dickler HB, et al. Indentifiction of analternative CTLA-4 ligand costimulatory for cell activation. Science. 1993, 262:905-909.
12 Linsley PS, Brady W, Grosmarie L, et al. Binding of the B cellactivation antigen B7 to CD28 costimulates T cell proliferation and interleakin 2 mRNA accumulation. J Exp Med. 1991,173:721-730.
13 Karandikar NJ, Vanderlugt CL, Walunas TL, et al. CRLA4:a negative regulator of autoimmune disease. J Exp Med, 1996, 184:783-792.
1 Shao Z, Chu Y, Zhang Y, et al. Treatment of severe aplastic anemia with an immunosuppressive agent plus recombinant human granulocyte macrophage colony stimulating factor and erythropoietin. Am J Hematol, 1998, 59:185-191.
2 Kaito K, Otsubo H, Usui N, et al. Th1/Th2 lymphocyte balance in patients with aplastic anemia. Rinsho Byori. 2004,52: 569-73.
3 Giannakoulas NC, Karakantza M, Theodorou GL, et al. Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients. Br J Haematol. 2004,124: 97-105.
4 Kurata H, Lee HJ, McClanahan T, et al. Friend of GATA is expressed in naive T_H cells and functions as a repressor of GATA-3 mediated Th2 cell development. J Immunol, 2002, 168: 4538-4545.
5 薛庆善主编．体外培养的原理与技术．北京：科学出版社，2001，593．
6 Shao Z, Clm Y, Zhang Y, et al. Treatment of severe aplastic anemia with an immunosuppressive agent plus recombinant human granulocyte macrophage colony-stimulating factor and erythropoietin. Am J Heanalnl, 1998, 59: 185-191.
7 Vlacicjewski JP, Hihhs JR, Anderson S, et.al. Bone marrow and peripheral blood lymphocyte phenotype in patients with bone marrow failure. Exp Hematol, 1994, 22: 1102-1110.
8 Nistico A, Young SS. Gamma-interferon gene expression in marrow of patients with aplastic: anemia. Ann Intern Med, 1994, 120: 463-469.
9 Nakao S, Yamaguchi M, Shiobara S, et al. Interferon-γ gene expression in unstimulated bone marrow mononuclear cells predicts a good response to cyclosporine therapy in aplastic anemia. Blood, 1992, 79: 2532-2535.
10 Laver J , Castro-Malaspina H, Kernan NA, et al. In vitro interferon gamma production by cultured T-cells in severe aplastic anemia: correlation with granulomonopoietic inhibition in patients who respond to antithymocyte globulin. Br J Haematol, 1988, 69:545-550.
11 Killick SB, Cox CV, Marsh JC, et al. Mechanisms of bone marrow progenitor cell apoptosis in aplastic anemia and the effect of antithymocyte glubin: examination of the role of the Fas-Fas-L interaction. Br J Haematol, 2000, 111: 1164-1169.
12 Maciejewski JP, Selleri C, Anderson S, et al. Fas antigen expression on CD34+ human marrow cells is induced by interferon-γand tumor necrosis factor-a and potentiates cytokine-mediated hematopoietic suppression in vitro. Blood, 1995, 85:3183-3190.
13 Maciejewski JP, Selleri C, Sato T, et al. Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anemia. Br J Haematol.1995; 91:245-252.
14 Romagnami S. Human Th1 and Th2 subsets: doubt no more. Immnnol Today, 1991, 12: 256-257.
15 Romagnami S. T cell subsets (Th1 versus Th2). Ann Allergy Asthma Immunol, 2000,85:9-18.
16 Haggqvist B, Hultman P. Effects of deviating the Th2 response in murine mercury induced autoimmunity towards a Thl response. Clin Exp Immuol, 2003, 134:202-209.
17 Dominguez-Garcia MV, Rodriguez-Moyado H. Cellular and biochemical mechanisms involved in physiopathgenesis of auto-immune thrombocytopenic purpura. Gac Med Mex, 2002, 138: 461-472.
18 Szabo SJ, Kim ST, Costa GL, et al. A novel transcription factor, T-bet, directs Thl linage commitment. Cell, 2000, 100: 655-669.
19 邢同京，章廉，主编．TH 类细胞极化群体的基础与临床．北京：军事医学科学出版社，2002，35-41．
20 Dorfman DM, Vanden-Elzen P, Weng AP, et al. Differential expression of T-bet, a T-box transcription factor required for T_H1 T-cell development, in peripheral T-cell lymphomas. Am J Clin Pathol, 2003, 120: 866-873.
21 Zheng WP, Flavell RA. The Transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CO_4~+ T cells. Cell, 1997, 89: 587-599.
22 Kaito K, Otsubo H, Usui N, et al. Th1/Th2 lymphocyte balance in patients with aplastic anemia. Rinsho Byori. 2004 ,52(7):569-73.
23 Giannakoulas NC, Karakantza M, Theodorou GL, et al. Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients. Br J Haematol. 2004, 124(1): 97-105.
1 Nakao S. Immune mechanism of aplastic anrmia. Int J Hematol 1997, 66:127-134.
2 Jaroslaw P, Maciejewski, Antomnio R, et al. Immune Pathophysiology of Aplastic Anemia.Inter J of Hematol, 2002, 76207-214.
3 Immunization with dendritic cells pulsed with tarnor extract increases survival of mice bearing intracranial gliomas. Ni HT, Spellman SR, Jean WC, et al. J Neurooncol. 2001, 51(1):1-9.
4 Cloned Dendritic Cells Can Present Exogenous Antigens on Both MHC Classs Ⅰ and Class Ⅱ Molecules. Zhenhai S, Glen R, Glenn D, et al. J Immunol. 1997, 158:2723-2730.
5 薛庆善主编．体外培养的原理与技术．北京：科学出版社，2001，201-201．
6 Young NS, Maciejewski J. The pathophysiology of acquired aplastic anemia. N Engl J Med. 1997, 336:1365—1372.
7 Vfolk A, Simon-Stoos K, Nami I, Concannon J, Mawe J, T anawattanacha-roen P, et al. A mouse model of immune-mediated aplastic anemia. Blood. 1998, 10(Suppl 1): 158a-159a.
8 Hoon Kook, Antonio M, Risitano WZ, et al. Changes in T-cell receptor VB repertoire in aplastic anemia: effects of different immunosuppressive regimens. Blood. 2002, 99:3668-3675.
9 Harding FA, McArthur JR, Gross JA, et al. CD28 mediated signaling costimulates murine T cells and prevents induction of anergy in T cell clones. Nature,1992;356:607-609.
10 Bugeon L, Dallman MJ. Costimulation of T cells. Am J Respir Crit Care Med, 2000, 162(4 Pt 2):S164-172.
11 Karandikar NJ, Vanderlugt CL, Walunas TL, et al. CRLA4:a negative regulator of autoimmune disease. J Exp Med, 1996, 184:783-792.
12 Balow JE, Boumpas DT, Austin HA, et al. New prospects for treatment of lupus nephritis. Semin Nephrol, 2000, 20: 32- 39.
13 Reynolds J, Tam FW, Chandraker A, et al. CD28-B7 blockade prevents the development of experimental auto-immune glomerulonephritis. J Clin Invest. 2000, 105: 643-651.
14 Perrin PJ, June CH, Maldonado JH, et al. Blockade of CD28 durinn in vitro activation of encephalitogenic T cells or after disease nset ameliorates experimental auto- immune encephalomyelitis. J Immunol. 1999, 163: 1704-1710.
15 Sato J, Asakura K, Murakami M, et al. Topical CTLA4- Ig suppresses ongoing mucosal immune response in pre-sensitized murine model of allergic rhinitis. Int Arch Allerny Immunol. 1999, 119:197-204.
16 Kaito K, Otsubo H, Usui N, et al. Th1/Th2 lymphocyte balance in patients with aplastic anemia. Rinsho Byori. 2004,52: 569-73.
17 Giannakoulas NC, Karakantza M, Theodorou GL, et al. Clinical relevance of balance between type 1 and type 2 immune responses of lymphocyte subpopulations in aplastic anaemia patients. Br J Haematol. 2004,124: 97-105.
18 Linslev PS, Wallace PM, Joneson J, et al. Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule. Science, 1992, 257(5071):792- 795.
1. Nakao S. Immune mechanism of aplastic anrmia.Int J Hematol 1997, 66:127-134.
2. Jaroslaw P, Maciejewski, Antomnio R, et al. Immune Pathophysiology of Aplastic Anemia.Inter J of Hematol, 2002, 76207-214.
3. Chiu KM, Knospe WH. Immunological mediated aplastic anemia mice: effects of varying the source and composition of donor cell. Exp Hematol, 1987, 15:269-275.
4. Melenhorst JJ, van Krieken JHJM, Dreef E, et al. T cell selectively infiltration bone marrow areas with residual haemopoiesis of patients with acquired aplastic anaemia. Br J Haematol, 1997, 99:527-519.
5. Viale M, Merli A, Bacigalupo A.Analysis at the clonal level of T-cell phenotype and function in sever aplastic anemia patients. Blood, 1991,78:1268-1274.
6. Macieje wski JP, Hibbs JR, Anderson S, et al. Blood marrow and peripheral blood lymphocyte phenotype in patients with bone marrow failure. Exp Hematol 1994, 22:1102-1110.
7. Hoon K, Weihua Z, Chen G, et al. Increased cytotoxic T cell with effector phenotype in aplastic anemia and myelodysplasia. Exp Hematol.2001, 29:1270-1277.
8. Hsu HC, Tsai WH, Chen LY, et al. Overproduction of inhibitory hematopoietic cytokines by lipoplysaccharide-activatde peripheral blood mononuclear cell in patients with aplastic anemia. Ann Hematol, 1995, 71:281-286.
9. Nistico A, Young NS, Gamma-interferon gene expression in the bone marrow of patients with aplastic anemia. Ann Intern Med, 1994, 120:463-469.
10.Nakao S, Yamaguchi M, Shiobara S, et al. Interferon-r gene expression in unstimulated bone marrow mononuclear cells predicts a good response to cycloporine therapy in aplastic anemia. Blood, 1992, 79:2532-2535.
11.Hiaoyuki T and Hiroshi Y. Type I and Type II T-cell Profiles in Aplastic Anemia and Refractory Anemia. American J of Hematol, 2000, 64:271-274.
12. Manz CY, Dietrich PY, Schnuriger V, et al. T-cellreceptor beta chain variability in bone marrow andperipheral blood in severe acquired aplastic anemia. Blood Cell S Mol Dis, 1997;23:110-121.
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