EAMG模型大鼠的实验研究
摘要
目的:从美国加利福尼亚电鳐的电器官中提取纯化乙酰胆碱受体(AchR),主动免疫Lewis大鼠,复制实验性自身免疫性重症肌无力(EAMG)动物模型。方法:参照国内外文献,利用免疫亲和层析技术提取纯化AchR,采用SDS聚丙烯酰胺凝胶电泳测蛋白纯度,观察免疫后大鼠每日表现,并于初次免疫后第4周、第8周作肌电图,实验结束时取血清,检测乙酰胆碱受体抗体AchR-ab,最后处死大鼠,电镜观察神经肌肉接头。结果:模型组大鼠在初次免疫注射后的第17天开始相继出现肌无力症状,并有三只迅速恶化,一周内死亡。模型组其它发病大鼠经肌电图证实,衰退程度均大于15%,而佐剂组与正常组大鼠均无表现。模型组AchR-ab高于佐剂组与正常组值,P<0.05。电镜观察模型组神经肌肉接头处(NMJ)突触前后膜有改变。
结论:从美国加利福尼亚电鳐的电器官中提取纯化乙酰胆碱受体,主动免疫Lewis大鼠,成功复制出EAMG模型,为中药治疗重症肌无力的研究提供了理想的动物模型。
Motive:Extracting pure AchR from American Torpedo California electric organ, immuning Lewis rats actively, duplicating EAMG animal model.Method:according to domestic and overseas literature, with immunoaffinity chromatography technology extracting pure AchR, adopting SDS polyacrylamide gel electrophoresis to detect purity of albumen, observing the immuned rats behaviour everyday, moreover making EMG in week 4 or 8 after the first immuning. When the experiment ended, the blood serum would be extracted to test AchR-ab. Finally executing the rats and observing neuromuscular junction with electron microscope.Result:The model group rats expressed myasthenia successively on the 17th day after the first immuning injection, besides there were 3 rats appearing worse and dying in a week. Affirmed by EMG, other rats of model group recession degree were exceeding 15% respectively.But neither did the adjuvant group and control group rats. The model group AchR-ab amount was higher than the adjuvant group and control group , p<0.05. Observing by electron microscope, we found that synapsis circa-membrane of the model group neuromuscular junction had changed.Conclusion: Extracting pure AchR from American Torpedo California electric organ, immuning Lewis rats actively, duplicating EAMG animal model successfully,Providing ideal animal model for the study of treating MG with Traditional Chinese medica.
结论:从美国加利福尼亚电鳐的电器官中提取纯化乙酰胆碱受体,主动免疫Lewis大鼠,成功复制出EAMG模型,为中药治疗重症肌无力的研究提供了理想的动物模型。
Motive:Extracting pure AchR from American Torpedo California electric organ, immuning Lewis rats actively, duplicating EAMG animal model.Method:according to domestic and overseas literature, with immunoaffinity chromatography technology extracting pure AchR, adopting SDS polyacrylamide gel electrophoresis to detect purity of albumen, observing the immuned rats behaviour everyday, moreover making EMG in week 4 or 8 after the first immuning. When the experiment ended, the blood serum would be extracted to test AchR-ab. Finally executing the rats and observing neuromuscular junction with electron microscope.Result:The model group rats expressed myasthenia successively on the 17th day after the first immuning injection, besides there were 3 rats appearing worse and dying in a week. Affirmed by EMG, other rats of model group recession degree were exceeding 15% respectively.But neither did the adjuvant group and control group rats. The model group AchR-ab amount was higher than the adjuvant group and control group , p<0.05. Observing by electron microscope, we found that synapsis circa-membrane of the model group neuromuscular junction had changed.Conclusion: Extracting pure AchR from American Torpedo California electric organ, immuning Lewis rats actively, duplicating EAMG animal model successfully,Providing ideal animal model for the study of treating MG with Traditional Chinese medica.
引文
[1] Im SH, Barchan D, Maiti PK, et al. Blockade of CD40 ligand suppresses chronic experimental myasthenia gravis by down-regulation of Th1 differentiation and up-regulation of CTLA-4. J Immunol, 2001, 166:6893-6898.
[2] Christadoss P, Poussin M, Deng C. Animal models of myasthenia gravis. Clin Immunol, 2000, 94: 75-87.
[3] Schimidt J., and Raftery M. A. Purification ofacetylcholine receptor from Torpedo californica electroplax by affinity chromatography. Biochemistry 197312:852—856.
[4] 徐浩鹏、孙曼霁、周廷冲 丁氏双鳍电鳐电器官烟碱样胆碱能受体的分离提纯 生物化学与生物物理学报 1984;1:50—55。
[5] 孙曼霁 烟碱样胆碱能受体的分离提纯 生物化学与生物物理进展 1982;6:55—59。
[6] 孙欣、龚潮樑、潘淑英、樊剑英、何其伟 丁氏双鳍电鳐电器官烟碱样乙酰胆碱受体某些生化性质的研究 动物学研究 1985;6(1):45—55。
[7] Schimidt J., and Raftery M. A. Purification of acetylcholine receptor from Torpedo californica electroplax by affinity chromatography. Biochemistry 1973 12:852—856.
[8] Claudio T., and Raftery M. A. Immunological comparison of acetylcholine receptor and subunits from species of Electric Ray. Arch. Biochem. Biophys. 1977 181:484—489.
[9] Lindstrom J., Einarson B., and Merlie J. Immunization of rats with polypeptide chains from Torpedo acetylcholine receptor causes an autoimmune response to receptors in rat muscle . Proc. Natl. Acad. Sci. U.S.A. 1978 75:769—733.
[10] Vandlen R. L., Wu W. CS., Eisenach J. C., and Raftery M. A. Studies of the composition of purified Torpedo californica acetylcholine receptor and of its subunits. Biochemistry 1979 18:1845—1854.
[ll]Hucho E., Layer P., Kiefer H. R., and Bandini G. Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica . Proc. Natl. Acad. Sci. U.S.A. 1976 73:2642—2628.
[12] Lennon VA, Lindstrom JM, Seybold ME . Experimental autoimmune my asthenia: a model of myasthenia gravis in rats and guinea pigs [J]. J Exp Med, 1975,141 (6): 1365-1375
[13] Lennam J AR, et al. Clinical Electromygraphy, ed2 Pitman, London, 1977:158
[14] Waser, P. G. et al. :Recent Advance in Receptor Chemistry (Ed. By Gualtier F. et al.)97 1979.
[15] Wilks S . Lectures on diseases of the nervous system delivered at guys hospital [M], 2~(nd). Philadelphia: P. Blakiston, Son &Co. 1883.
[16] Vinken PJ, Bruya GW. Handbook of clinical neurology. New York, Northe Holland Company , 1979 : 95-122.
[17] Dale HH, Feldberg W, Vogt M. Release of acetylcholine at voluntary motor nerve endings[J]. J Physiol (Lond) , 1936, 86: 353-380.
[18] Walker MB. Treatment of myasthenia gravis with physostigmin [J]. Lancet, 1934, 1: 1200-1201.
[19] Blalock A, Mason MF, Morgan HJ, et a 1 . Myasthenia gravis and tumors of the thymic region. Report of a case I which the tumor was removed[J]. Ann Surg, 1939, 110: 544-561
[20] Harvey AM , Lilienthal JL JR, Talbot SA. Observations on the nature of myasthenia gravis : the phenomena of facilitation and depression of neuromuscular transmission [J]. Bull Johns Hopkins Hosp, 1941, 69: 547-565.
[2l]Whiting P, Lindstrom J, Purification and characterization of anticotinic acetylcholine receptor from rat brain. Proc Natl Acad Sci USA, 1987, 84: 595.
[22] Simpson JA. Myasthenia gravi : a new hypothesis [J]. Scott Med J, 1960, 4: 419-436.
[23] Vinken PJ, Bruya GW. Handbook of clinical neurology. New York. Northe Holland Company. 1979: 95-122.
[24] Patrick J, Lindstrom JM, Culp B, et al. Studies on purefied eel acetycholine receptor andanti-acetylcholine receptor anti-body. Proc Nat Acad Sci. 1973, 70: 3331.
[25] Fambrough DM, Drachman DB, Satyamurti S. Neuromusculer junction in myasthenia grvis: decreused acetylcheline receptors [J]. Science, 1973, 182: 293-295.
[26] Engel AG, Lambert EH, Howard FM. Immune complexes (IgG and C3) at the motor end-plate in myasthenia gravis. Mayoclin Proc. 1977, 52: 276.
[27] Grob D, Simpson D, Metsumoto H, et al. Treatment ofmyasthenia gravis by immunoadsorption of plasma. Neurol. 1995, 45: 338.
[28] Scelsa S, Frost M, Valderrama R, et al, Prevention and treatment of experimental myasthenia gravis with anti-rabbit thymocyte serum and gammaglobulin in rabbits. Pathobiology. 1995, 63(3):168-174.
[29] 殷伟、乔健、吕传真 重症肌无力实验模型中胸腺细胞凋亡及其机制的研究 上海免疫学杂志 2000,20(1)44-46
[30] 梁丽云 马存根 张光先 肖保国 TGF-β上调在Wistar大鼠抑制EAMG发生中起重要作用大同医专学报 1999,19(2):4-7。
[31] Burges J. Vincent A, Molenaar PC, et al. Passive transfer of seronegative myasthenia gravis to mice, Muscle Nerve, 1994, 17(12):1396-1400
[32] Wang HB, Shi FD, Li H, et al. Anti-CTLA-4 antibody treatment triggers determinant spreading and enhances murine myasthenia gravis. J Immunol, 2001, 166(10):6430-6436
[33] Paas-Rozner M, Sela M, Mozes E. The nature of the active suppression of responses associated with experimental autoimmune myasthenia gravis by a dual altered peptide ligand administered by different routes. Proc Natl Acad Sci U S A, 2001, 98(22);12642-12647
[34] Paas-Rozner M, Dayan M, Paas Y, et al. Oral administration of asual analog of two myasthenogenic T cell epitopes down-regulates experimental autoimmune myasthenia gravis in mice, Proc Natl acad sci U S A, 2000, 97(5):2168-2173
[35] Oshima M, Pachner AR, Atassi MZ. Molprofile of the regions of acetylcholine receptor alpha chain recognized by T-lymphocytes and by antibodies in EAMG-susceptible and non-susceptible mouse strains after different periods of immunization with the receptor. Immunol, 1994, 31(11):833-843.
[36] 韩涛 重肌灵对EAMG大鼠免疫调节作用的实验研究 北京中医药大学学报 2003,26(3)
[37] 王旭丹 重肌灵片对实验性重症肌无力的影响 上海免疫学杂志 2003,23(5)
[2] Christadoss P, Poussin M, Deng C. Animal models of myasthenia gravis. Clin Immunol, 2000, 94: 75-87.
[3] Schimidt J., and Raftery M. A. Purification ofacetylcholine receptor from Torpedo californica electroplax by affinity chromatography. Biochemistry 197312:852—856.
[4] 徐浩鹏、孙曼霁、周廷冲 丁氏双鳍电鳐电器官烟碱样胆碱能受体的分离提纯 生物化学与生物物理学报 1984;1:50—55。
[5] 孙曼霁 烟碱样胆碱能受体的分离提纯 生物化学与生物物理进展 1982;6:55—59。
[6] 孙欣、龚潮樑、潘淑英、樊剑英、何其伟 丁氏双鳍电鳐电器官烟碱样乙酰胆碱受体某些生化性质的研究 动物学研究 1985;6(1):45—55。
[7] Schimidt J., and Raftery M. A. Purification of acetylcholine receptor from Torpedo californica electroplax by affinity chromatography. Biochemistry 1973 12:852—856.
[8] Claudio T., and Raftery M. A. Immunological comparison of acetylcholine receptor and subunits from species of Electric Ray. Arch. Biochem. Biophys. 1977 181:484—489.
[9] Lindstrom J., Einarson B., and Merlie J. Immunization of rats with polypeptide chains from Torpedo acetylcholine receptor causes an autoimmune response to receptors in rat muscle . Proc. Natl. Acad. Sci. U.S.A. 1978 75:769—733.
[10] Vandlen R. L., Wu W. CS., Eisenach J. C., and Raftery M. A. Studies of the composition of purified Torpedo californica acetylcholine receptor and of its subunits. Biochemistry 1979 18:1845—1854.
[ll]Hucho E., Layer P., Kiefer H. R., and Bandini G. Photoaffinity labeling and quaternary structure of the acetylcholine receptor from Torpedo californica . Proc. Natl. Acad. Sci. U.S.A. 1976 73:2642—2628.
[12] Lennon VA, Lindstrom JM, Seybold ME . Experimental autoimmune my asthenia: a model of myasthenia gravis in rats and guinea pigs [J]. J Exp Med, 1975,141 (6): 1365-1375
[13] Lennam J AR, et al. Clinical Electromygraphy, ed2 Pitman, London, 1977:158
[14] Waser, P. G. et al. :Recent Advance in Receptor Chemistry (Ed. By Gualtier F. et al.)97 1979.
[15] Wilks S . Lectures on diseases of the nervous system delivered at guys hospital [M], 2~(nd). Philadelphia: P. Blakiston, Son &Co. 1883.
[16] Vinken PJ, Bruya GW. Handbook of clinical neurology. New York, Northe Holland Company , 1979 : 95-122.
[17] Dale HH, Feldberg W, Vogt M. Release of acetylcholine at voluntary motor nerve endings[J]. J Physiol (Lond) , 1936, 86: 353-380.
[18] Walker MB. Treatment of myasthenia gravis with physostigmin [J]. Lancet, 1934, 1: 1200-1201.
[19] Blalock A, Mason MF, Morgan HJ, et a 1 . Myasthenia gravis and tumors of the thymic region. Report of a case I which the tumor was removed[J]. Ann Surg, 1939, 110: 544-561
[20] Harvey AM , Lilienthal JL JR, Talbot SA. Observations on the nature of myasthenia gravis : the phenomena of facilitation and depression of neuromuscular transmission [J]. Bull Johns Hopkins Hosp, 1941, 69: 547-565.
[2l]Whiting P, Lindstrom J, Purification and characterization of anticotinic acetylcholine receptor from rat brain. Proc Natl Acad Sci USA, 1987, 84: 595.
[22] Simpson JA. Myasthenia gravi : a new hypothesis [J]. Scott Med J, 1960, 4: 419-436.
[23] Vinken PJ, Bruya GW. Handbook of clinical neurology. New York. Northe Holland Company. 1979: 95-122.
[24] Patrick J, Lindstrom JM, Culp B, et al. Studies on purefied eel acetycholine receptor andanti-acetylcholine receptor anti-body. Proc Nat Acad Sci. 1973, 70: 3331.
[25] Fambrough DM, Drachman DB, Satyamurti S. Neuromusculer junction in myasthenia grvis: decreused acetylcheline receptors [J]. Science, 1973, 182: 293-295.
[26] Engel AG, Lambert EH, Howard FM. Immune complexes (IgG and C3) at the motor end-plate in myasthenia gravis. Mayoclin Proc. 1977, 52: 276.
[27] Grob D, Simpson D, Metsumoto H, et al. Treatment ofmyasthenia gravis by immunoadsorption of plasma. Neurol. 1995, 45: 338.
[28] Scelsa S, Frost M, Valderrama R, et al, Prevention and treatment of experimental myasthenia gravis with anti-rabbit thymocyte serum and gammaglobulin in rabbits. Pathobiology. 1995, 63(3):168-174.
[29] 殷伟、乔健、吕传真 重症肌无力实验模型中胸腺细胞凋亡及其机制的研究 上海免疫学杂志 2000,20(1)44-46
[30] 梁丽云 马存根 张光先 肖保国 TGF-β上调在Wistar大鼠抑制EAMG发生中起重要作用大同医专学报 1999,19(2):4-7。
[31] Burges J. Vincent A, Molenaar PC, et al. Passive transfer of seronegative myasthenia gravis to mice, Muscle Nerve, 1994, 17(12):1396-1400
[32] Wang HB, Shi FD, Li H, et al. Anti-CTLA-4 antibody treatment triggers determinant spreading and enhances murine myasthenia gravis. J Immunol, 2001, 166(10):6430-6436
[33] Paas-Rozner M, Sela M, Mozes E. The nature of the active suppression of responses associated with experimental autoimmune myasthenia gravis by a dual altered peptide ligand administered by different routes. Proc Natl Acad Sci U S A, 2001, 98(22);12642-12647
[34] Paas-Rozner M, Dayan M, Paas Y, et al. Oral administration of asual analog of two myasthenogenic T cell epitopes down-regulates experimental autoimmune myasthenia gravis in mice, Proc Natl acad sci U S A, 2000, 97(5):2168-2173
[35] Oshima M, Pachner AR, Atassi MZ. Molprofile of the regions of acetylcholine receptor alpha chain recognized by T-lymphocytes and by antibodies in EAMG-susceptible and non-susceptible mouse strains after different periods of immunization with the receptor. Immunol, 1994, 31(11):833-843.
[36] 韩涛 重肌灵对EAMG大鼠免疫调节作用的实验研究 北京中医药大学学报 2003,26(3)
[37] 王旭丹 重肌灵片对实验性重症肌无力的影响 上海免疫学杂志 2003,23(5)