重症肌无力患者CD40配体表达的研究
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摘要
目的：(1)研究重症肌无力(MG)患者CD40配体(CD40L)的表达，探讨CD40L在MG中的作用。(2)探讨MG患者CD40L表达的信号传导途径。
方法：研究对象为25例MG患者，分为急性期组13例，非急性期组12例，设正常对照组15例。分离血中单个核细胞，用流式细胞仪检测CD40L阳性细胞率，应用RT—PCR方法检测单个核细胞CD40L mRNA的表达。再分组用刺激剂刺激进行单个核细胞培养：①PMA组：即用PMA和A23187刺激；②PHA组：即用PHA刺激。培养后用流式细胞仪检测CD40L阳性细胞率及RT—PCR法检测单个核细胞CD40LmRNA，并与刺激前作比较，同时还进行急性期组、非急性期组和正常对照组之间的比较。急性期组先加PKC抑制剂BLM后再加PHA刺激培养(BLM组)，然后检测CD40L阳性细胞率及CD40LmRNA(方法如前述)，并与PMA组、PHA组及纯培养组作比较分析。
结果：(1)MG急性期组CD40L阳性细胞率和CD40L mRNA均比正常对照组及非急性期组显著增高(均为P＜0.01)，而非急性期组CD40L阳性细胞率和CD40LmRNA与正常对照组相比无显著性差异(P＞0.05)；(2)经PMA或PHA刺激后，
广西医科大学硕士研究生学位论文
不但急性期组 CD40L阳性细胞率和 CD40 Lm叫A仍比非急性
期组和正常对照组显著增高（均为P＜0．01卜而且非急性期组上
述指标也比正常对照组显著增高呼 刀1）；门）MG急性期
BLM组 CD40L阳性细胞率和 CD40L mRNA与纯培养组无明显
差别（Ptoo．05），而显著低 于PMA组和P枷组（Pwto．01）。
结论：（）CD40L的高表达是MG发生和加重的因素八2）
在MG中，CD40L的表达可能是依赖于PKC活性介导的T细
胞活化途径。
Objectives: (l)To investigate the role of CD40 and CD40 ligand in the pathogenesis of myasthenia gravis (MG) , the CD40L expression on the peripheral blood mononuclear cells of patients with MG was tested. (2) To investigate the signal transduction pathway involved in CD40L expression of patients with MG.
Methods: 25 patients with MG included 13 acute patients and 12 non-acute patients. 15 healthy people was acted as control group. Blood of patients and normal controls was collected. Mononuclear cells in peripheral blood(PBMC) were isolated and cultured in the medium(RPMI-1640) supplemented with PMA and A23187 or PHA. After 6 hours, cell samples were collected and CD40L expression was tested by flow cytometry and reverse transcription-polymerase chain reaction(RT-RCR) compared with that of them before activation. Except for PMA and A23187 or PHA treatment, a specific inhibitor of PKC, bisindolylmaleimide(BLM),was added to the cells before exposure to PHA in patients with acute MG. Meanwhile, the medium without activator in PBMC cultures was used in acute MG .Cells in
different culture medium were collected and CD40L expression was tested .
Results: (1) CD40L expression on PBMC in patients with acute MG was increased markedly compared with that of patients with non-acute MG and normal controls before activation, but there were no significantly statistics differences between patients with non-acute MG and normal controls. (2)CD40L expression of patients with acute MG and non-acute MG were increased significantly in contrast to that of normal controls after activation with PHA or PMA CD40L expression of patients with acute MG still increased markedly compared with that of patients with non-acute MG. (3)CD40L expression of patient with acute MG after activation with PHA was not different from that after activation with PMA, but there was increased markedly in contrast to that of the status of medium without activator. CD40L expression in the presence of the PKC inhibitor was no significantly difference compared with that of the status of medium without activator.
Conclusions: (1) Hyperexpression of CD40L is correlated with the clinical progression of MG. (2) CD40L expression in MG is mediated by PKC activity.
方法：研究对象为25例MG患者，分为急性期组13例，非急性期组12例，设正常对照组15例。分离血中单个核细胞，用流式细胞仪检测CD40L阳性细胞率，应用RT—PCR方法检测单个核细胞CD40L mRNA的表达。再分组用刺激剂刺激进行单个核细胞培养：①PMA组：即用PMA和A23187刺激；②PHA组：即用PHA刺激。培养后用流式细胞仪检测CD40L阳性细胞率及RT—PCR法检测单个核细胞CD40LmRNA，并与刺激前作比较，同时还进行急性期组、非急性期组和正常对照组之间的比较。急性期组先加PKC抑制剂BLM后再加PHA刺激培养(BLM组)，然后检测CD40L阳性细胞率及CD40LmRNA(方法如前述)，并与PMA组、PHA组及纯培养组作比较分析。
结果：(1)MG急性期组CD40L阳性细胞率和CD40L mRNA均比正常对照组及非急性期组显著增高(均为P＜0.01)，而非急性期组CD40L阳性细胞率和CD40LmRNA与正常对照组相比无显著性差异(P＞0.05)；(2)经PMA或PHA刺激后，
广西医科大学硕士研究生学位论文
不但急性期组 CD40L阳性细胞率和 CD40 Lm叫A仍比非急性
期组和正常对照组显著增高（均为P＜0．01卜而且非急性期组上
述指标也比正常对照组显著增高呼 刀1）；门）MG急性期
BLM组 CD40L阳性细胞率和 CD40L mRNA与纯培养组无明显
差别（Ptoo．05），而显著低 于PMA组和P枷组（Pwto．01）。
结论：（）CD40L的高表达是MG发生和加重的因素八2）
在MG中，CD40L的表达可能是依赖于PKC活性介导的T细
胞活化途径。
Objectives: (l)To investigate the role of CD40 and CD40 ligand in the pathogenesis of myasthenia gravis (MG) , the CD40L expression on the peripheral blood mononuclear cells of patients with MG was tested. (2) To investigate the signal transduction pathway involved in CD40L expression of patients with MG.
Methods: 25 patients with MG included 13 acute patients and 12 non-acute patients. 15 healthy people was acted as control group. Blood of patients and normal controls was collected. Mononuclear cells in peripheral blood(PBMC) were isolated and cultured in the medium(RPMI-1640) supplemented with PMA and A23187 or PHA. After 6 hours, cell samples were collected and CD40L expression was tested by flow cytometry and reverse transcription-polymerase chain reaction(RT-RCR) compared with that of them before activation. Except for PMA and A23187 or PHA treatment, a specific inhibitor of PKC, bisindolylmaleimide(BLM),was added to the cells before exposure to PHA in patients with acute MG. Meanwhile, the medium without activator in PBMC cultures was used in acute MG .Cells in
different culture medium were collected and CD40L expression was tested .
Results: (1) CD40L expression on PBMC in patients with acute MG was increased markedly compared with that of patients with non-acute MG and normal controls before activation, but there were no significantly statistics differences between patients with non-acute MG and normal controls. (2)CD40L expression of patients with acute MG and non-acute MG were increased significantly in contrast to that of normal controls after activation with PHA or PMA CD40L expression of patients with acute MG still increased markedly compared with that of patients with non-acute MG. (3)CD40L expression of patient with acute MG after activation with PHA was not different from that after activation with PMA, but there was increased markedly in contrast to that of the status of medium without activator. CD40L expression in the presence of the PKC inhibitor was no significantly difference compared with that of the status of medium without activator.
Conclusions: (1) Hyperexpression of CD40L is correlated with the clinical progression of MG. (2) CD40L expression in MG is mediated by PKC activity.
引文
[1] Durie FH, Foy T M, Masters S R, et al. The role of CD40 in the regulation of humoral and cell-mediated immunity. Immunol Today 1994,15:406
[2] Stout RD, and Suteles J. The many role of CD40 in cell mediated in flammatory responses. Immunol Today 1996,17:487
[3] Grewal I and Flavell R A, The CD40 ligcand:at the center of immune universe? Immunol 1997,16:59
[4] Kennedy, M K, Mohter KM, Shanebeek K D, et al. Induction of B cell costimulatory function by recombinant murine CD40 ligand. Eur J Immunol 1994,24:16
[5] Koy M, Aruffo A, Edbetter J, et al. Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune resgonses. Eur J Immunol 1995,25:596
[6] Stout R D, Suttles J, Xu J, et al. Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice. J Immunol
1996,156:8
[7] Grewal I S, Su J, Flavell R A, et al. Impairment of antigen-specific T-cell priming in mice lacking CD40 ligand.Nature 1995,278:617
[8] Durie F H, Fava R A, Foy T M, Prevention of collagen-induced arthritis with an antibody to gp39, The ligand for CD40． science 1993,261:1328
[9] Griggs N D, Agersborg SS, Noelle R J, et al. The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of s elf-reactive T cells.J Exp Med 1996,183:801
[10] Gerritse K, Laman J D, Noelle R J,et al. CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Natl Acad Sci USA 1996,93:2499
[11] Grewal I S, Foellmer H G, Grewal K D, et al. Requirement for CD40 ligand in Costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 1996, 273:1864
[12] Mohan C, Shi Y, Laman J D, et al. Interaction between CD40 and its ligand 9p39 in the development of murine lupus nephritis. J Immunol 1995,154:1470
[13] Koshy M, berger D, Crow M K, Increased expression of CD40 ligand on systemic lupus. J Clin Invest 1997,98:826
[14] Carayanniotis G, Masters S R, Noelle R J, et al. suppression of murine thyroiditis via blockade of the CD40-CD40L interaction.Immunology 1997,90:421
[15] Balasa B, Krahl T, Patstsne J, et al. CD40 ligand-CD40 interactions are necessary for the initiation of insulitis and diabetes in nonobase diabetic mice. J Immunol 1997,159:4620
[16] Shi F D, He B, Li H, et al. Differential requirements for CD28 and CD40 ligand in the induction of experimental autoimmune myasthenia gravis. Eur J Immunol 1998,28:3587
[17] 孙宏,国红,许贤豪等,重症肌无力患者外周血乙酰胆碱 受体特异性T细胞SLL-2R、IFN-γ、IL-4的表达和转录。 中华神经科杂志 1998,31(2) :79
[18] Desal-Metha A, Lu L, Ramsey-Goldman, et al.Hyperexpression of CD40 ligand by B and T cells in Human Lupus and its role in pathogenic autoantibody production. J Clin Invest 1996,97:2063
[19] Lindstrom J M, Seybold M E, Lennon V A, et al. Antibody to acetylcholine receptor in myasthenia gravis:prevalence, Clinical correlates, and diagnostic Value. Neurology 1976,26:1054
[20] lefvert A K, Haman and experimental myasthenia gravis In: Wiley,ed.Autoimmunity:physiology and disease.Stockholm:Wiley-liss 1994,267
[21] Rapport B, Portolano S, Mclachlan S M, et al. Combinational libraries:new insight into organ-specific autoantibodied. Immunol Today 1995,16:43
[22] 莫雪安，郑金瓯，马朝桂。重症肌无力自身抗体、T淋巴细 胞亚群及其临床意义的探讨。广西医科大学学报 1998,15:20
[23] Jrolick K, Urso O, Analysis of helper-T-cell function by acetylcholine cell lines of defined AchR-subunit specificity. Cell Inmunol 1987,105:75
[24] Banchereau JF, Bazan D, Blanchard F, et al. The CD40 antigen and its ligand. Ann Rev Immunol 1994,12:881
[25] Grewal I S, Flaveil RA.CD40 and CD154 in cell-mediated immunity. Annv Rev Immunol 1998,16:111
[26] FoY T M, Aruffo A, Bajorath J. et al. Immune regulation by CD40 and its ligand 9p39． Annv Rev Immunol 1996,14:591
[27] Mclellan A D, Sorg R V, Willians L A, et al. Human dendritic cells activate T lymphocytes via a CD40:CD40 ligand-dependent pathway. Eur J Immunol 1996,26:1204
[28] Peng X H, Kasran A, Warmerdan PAM, et al. Accessory signaling by CD40 for T cell activation:induction of Thl and Th2 cytokines and synergy with interleukin-12 for interferon-r production. Eur J Immunol 1996,26:1621
[29] Kennedy M K, Picha K S, Fanslow W C, et al. CD40/CD40L
interactions are required for T cell dependent production of interleukin-12 by mouse macrophages. Eur J Immunol 1996,26:370
[30] Im SH, Barchan D, Maiti PK, et al, Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Thl differentiation and up-regulation of CTLA-4． J Immunol 2001,166:6893
[31] 何球藻，吴厚升，曹雪涛(主编)。细胞与分子免疫学1997, 第一版 P201-206
[32] Toullec D, Pianetti p, Coset H, et al.The bisindolylmaleimide G F 10923X is a potent and selective inhibitor of prtein kinasec.J Biol Chem 1991,266:15771
[2] Stout RD, and Suteles J. The many role of CD40 in cell mediated in flammatory responses. Immunol Today 1996,17:487
[3] Grewal I and Flavell R A, The CD40 ligcand:at the center of immune universe? Immunol 1997,16:59
[4] Kennedy, M K, Mohter KM, Shanebeek K D, et al. Induction of B cell costimulatory function by recombinant murine CD40 ligand. Eur J Immunol 1994,24:16
[5] Koy M, Aruffo A, Edbetter J, et al. Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune resgonses. Eur J Immunol 1995,25:596
[6] Stout R D, Suttles J, Xu J, et al. Impaired T cell-mediated macrophage activation in CD40 ligand-deficient mice. J Immunol
1996,156:8
[7] Grewal I S, Su J, Flavell R A, et al. Impairment of antigen-specific T-cell priming in mice lacking CD40 ligand.Nature 1995,278:617
[8] Durie F H, Fava R A, Foy T M, Prevention of collagen-induced arthritis with an antibody to gp39, The ligand for CD40． science 1993,261:1328
[9] Griggs N D, Agersborg SS, Noelle R J, et al. The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of s elf-reactive T cells.J Exp Med 1996,183:801
[10] Gerritse K, Laman J D, Noelle R J,et al. CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Natl Acad Sci USA 1996,93:2499
[11] Grewal I S, Foellmer H G, Grewal K D, et al. Requirement for CD40 ligand in Costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 1996, 273:1864
[12] Mohan C, Shi Y, Laman J D, et al. Interaction between CD40 and its ligand 9p39 in the development of murine lupus nephritis. J Immunol 1995,154:1470
[13] Koshy M, berger D, Crow M K, Increased expression of CD40 ligand on systemic lupus. J Clin Invest 1997,98:826
[14] Carayanniotis G, Masters S R, Noelle R J, et al. suppression of murine thyroiditis via blockade of the CD40-CD40L interaction.Immunology 1997,90:421
[15] Balasa B, Krahl T, Patstsne J, et al. CD40 ligand-CD40 interactions are necessary for the initiation of insulitis and diabetes in nonobase diabetic mice. J Immunol 1997,159:4620
[16] Shi F D, He B, Li H, et al. Differential requirements for CD28 and CD40 ligand in the induction of experimental autoimmune myasthenia gravis. Eur J Immunol 1998,28:3587
[17] 孙宏,国红,许贤豪等,重症肌无力患者外周血乙酰胆碱 受体特异性T细胞SLL-2R、IFN-γ、IL-4的表达和转录。 中华神经科杂志 1998,31(2) :79
[18] Desal-Metha A, Lu L, Ramsey-Goldman, et al.Hyperexpression of CD40 ligand by B and T cells in Human Lupus and its role in pathogenic autoantibody production. J Clin Invest 1996,97:2063
[19] Lindstrom J M, Seybold M E, Lennon V A, et al. Antibody to acetylcholine receptor in myasthenia gravis:prevalence, Clinical correlates, and diagnostic Value. Neurology 1976,26:1054
[20] lefvert A K, Haman and experimental myasthenia gravis In: Wiley,ed.Autoimmunity:physiology and disease.Stockholm:Wiley-liss 1994,267
[21] Rapport B, Portolano S, Mclachlan S M, et al. Combinational libraries:new insight into organ-specific autoantibodied. Immunol Today 1995,16:43
[22] 莫雪安，郑金瓯，马朝桂。重症肌无力自身抗体、T淋巴细 胞亚群及其临床意义的探讨。广西医科大学学报 1998,15:20
[23] Jrolick K, Urso O, Analysis of helper-T-cell function by acetylcholine cell lines of defined AchR-subunit specificity. Cell Inmunol 1987,105:75
[24] Banchereau JF, Bazan D, Blanchard F, et al. The CD40 antigen and its ligand. Ann Rev Immunol 1994,12:881
[25] Grewal I S, Flaveil RA.CD40 and CD154 in cell-mediated immunity. Annv Rev Immunol 1998,16:111
[26] FoY T M, Aruffo A, Bajorath J. et al. Immune regulation by CD40 and its ligand 9p39． Annv Rev Immunol 1996,14:591
[27] Mclellan A D, Sorg R V, Willians L A, et al. Human dendritic cells activate T lymphocytes via a CD40:CD40 ligand-dependent pathway. Eur J Immunol 1996,26:1204
[28] Peng X H, Kasran A, Warmerdan PAM, et al. Accessory signaling by CD40 for T cell activation:induction of Thl and Th2 cytokines and synergy with interleukin-12 for interferon-r production. Eur J Immunol 1996,26:1621
[29] Kennedy M K, Picha K S, Fanslow W C, et al. CD40/CD40L
interactions are required for T cell dependent production of interleukin-12 by mouse macrophages. Eur J Immunol 1996,26:370
[30] Im SH, Barchan D, Maiti PK, et al, Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Thl differentiation and up-regulation of CTLA-4． J Immunol 2001,166:6893
[31] 何球藻，吴厚升，曹雪涛(主编)。细胞与分子免疫学1997, 第一版 P201-206
[32] Toullec D, Pianetti p, Coset H, et al.The bisindolylmaleimide G F 10923X is a potent and selective inhibitor of prtein kinasec.J Biol Chem 1991,266:15771