PPAR-γ激动剂治疗实验性自身免疫性脑脊髓炎的实验研究
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摘要
目的 多发性硬化(multiple sclerosis,MS)是一种病因未明的中枢神经系统(central nervous system ,CNS)慢性炎症性脱髓鞘疾病,临床上缺乏确切有效的治疗手段。本研究通过实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis ,EAE)动物模型的建 立 , 应 用 过 氧 化 物 酶 体 增 殖 物 激 活 剂 受 体 -γ (peroxisomeproliferator-actived receptor-γ,PPAR-γ) 激动剂罗格列酮和非甾体类抗炎药(non-steroidal antiinflammatory drugs, NSAIDs)布洛芬治疗 EAE大鼠,观察致病动物的临床表现、发病率,通过检测前炎症细胞因子肿瘤坏死因子-α (tumor necrosis factor-α ,TNF-α )和钙激活中性蛋白酶(Calpain)的表达水平,探讨 PPAR-γ 激动剂罗格列酮和非甾体类抗炎药布洛芬在 EAE 中的疗效及其作用机制,为扭转临床上多发性硬化治疗的困难局面,提供新的方法和实验依据。
方法 ⑴ 以豚鼠脑脊髓匀浆加完全弗氏佐剂(complete Freund'sadjuvant,CFA)作为抗原,给予 Wistar 大鼠足垫皮下注射,另外在足背皮内注射百日咳杆菌,建立大鼠 EAE 模型。⑵ 将实验动物随机分成四组,正常对照组、EAE 模型组、罗格列酮治疗组和布洛芬治疗组,每组 8 只。从免疫后第 2 天开始,给予动物药物干预。罗格列酮的剂量为 4mg·kg-1·d-1;布洛芬的剂量为 100mg·kg-1·d-1灌胃治疗;其余两组用等量生理盐水替代。⑶ 观察所有动物的临床表现,按 Koh 评分标准评分;在免疫后第 18 天,处死所有动物,留取脑标本。部分做 HE 染色,其
Objective: This study’s objectives were to ⑴ investigate theefficacy of PPAR-γ agonist Rosiglitazone and non-steroidalantiinflammatory drugs (NSAIDs) Ibuprofen and their mode of action inexperimental autoimmune Encephalomyelitis (EAE) , which is a model tosimulate inflammatory aspects of Multiple sclerosis. ⑵ characterize theclinical features, the incidence of this disease in Wistar rats with EAE toevaluate dependability of the animal model. ⑶ determine the level of theexpression of TNF-α mRNA and Calpain further to evaluate efficacy andmechanism of the two agents and provide some experimental evidences forclinical therapies in MS.
Methods: All of the experimental Wistar rats were inoculatedintradermal injection with the emulsion homogenate contained spinal cordof guinea pig and complete Freund’s adjuvant in feet. The experimental ratswere assigned to four groups randomly,the normal control group, the EAEmodel group, Rosiglitazone treated group and Ibuprofen treated group.There are 8 rats in each group. Respectively, Rats were given the twoagents by oral gavage from the first day to 18th day after initialimmunization. The dosage of rosiglitazone was 4mg·kg-1·d-1, Ibuprofen 100mg·kg-1·d-1. The rats in normal control group were treated in stead of equalnormal saline (N.S). We have investigated the clinical signs and scores ofthe animals on the scale of koh's standard. 18th day after initialimmunization, All rats were killed and detected the expression of TNF-α
mRNA and Calpain by the RT-PCR and Western Blot.
Results: The animal model we induced showed typical clinicalcharacters and the pathological changes EAE. The expression of TNF-αmRNA was significantly increased in EAE group compare with normalcontrol group (P<0.01). The expression of TNF-α mRNA were significantlydecreased in Rosiglitazone treated group and Ibuprofen treated groupcompare with EAE group (P<0.01). The transcriptional expression ofm-Calpain as examined by RT-PCR was not significantly increased in EAEgroup(P > 0.05). However, the expression of Calpain protein wassignificantly increased in EAE rats compare with normal control group(P<0.01). The efficacy between rosiglitazone and Ibuprofen was nosignificant different.
Conclusions: 1)The method of establishing the model of EAE wasstable and reliable in our study. 2)PPAR-γ agonists rosiglitazone andnon-steroidal antiinflammatory drugs (NSAIDs) Ibuprofen can improve theclinical symptoms and signs of EAE in rats, and down modulate the levelof the expression of TNF-αmRNA and Calpain protein. These resultssuggested that orally administered PPAR-γ agonists rosiglitazone andIbuprofen could provide therapeutic benefit in EAE or MS. 3)Our findingssuggested that Calpain played a major role in demyelination in EAE. Thelevel of the expression of Calpain protein indicates the efficacy of thetherapeutic drugs.
方法 ⑴ 以豚鼠脑脊髓匀浆加完全弗氏佐剂(complete Freund'sadjuvant,CFA)作为抗原,给予 Wistar 大鼠足垫皮下注射,另外在足背皮内注射百日咳杆菌,建立大鼠 EAE 模型。⑵ 将实验动物随机分成四组,正常对照组、EAE 模型组、罗格列酮治疗组和布洛芬治疗组,每组 8 只。从免疫后第 2 天开始,给予动物药物干预。罗格列酮的剂量为 4mg·kg-1·d-1;布洛芬的剂量为 100mg·kg-1·d-1灌胃治疗;其余两组用等量生理盐水替代。⑶ 观察所有动物的临床表现,按 Koh 评分标准评分;在免疫后第 18 天,处死所有动物,留取脑标本。部分做 HE 染色,其
Objective: This study’s objectives were to ⑴ investigate theefficacy of PPAR-γ agonist Rosiglitazone and non-steroidalantiinflammatory drugs (NSAIDs) Ibuprofen and their mode of action inexperimental autoimmune Encephalomyelitis (EAE) , which is a model tosimulate inflammatory aspects of Multiple sclerosis. ⑵ characterize theclinical features, the incidence of this disease in Wistar rats with EAE toevaluate dependability of the animal model. ⑶ determine the level of theexpression of TNF-α mRNA and Calpain further to evaluate efficacy andmechanism of the two agents and provide some experimental evidences forclinical therapies in MS.
Methods: All of the experimental Wistar rats were inoculatedintradermal injection with the emulsion homogenate contained spinal cordof guinea pig and complete Freund’s adjuvant in feet. The experimental ratswere assigned to four groups randomly,the normal control group, the EAEmodel group, Rosiglitazone treated group and Ibuprofen treated group.There are 8 rats in each group. Respectively, Rats were given the twoagents by oral gavage from the first day to 18th day after initialimmunization. The dosage of rosiglitazone was 4mg·kg-1·d-1, Ibuprofen 100mg·kg-1·d-1. The rats in normal control group were treated in stead of equalnormal saline (N.S). We have investigated the clinical signs and scores ofthe animals on the scale of koh's standard. 18th day after initialimmunization, All rats were killed and detected the expression of TNF-α
mRNA and Calpain by the RT-PCR and Western Blot.
Results: The animal model we induced showed typical clinicalcharacters and the pathological changes EAE. The expression of TNF-αmRNA was significantly increased in EAE group compare with normalcontrol group (P<0.01). The expression of TNF-α mRNA were significantlydecreased in Rosiglitazone treated group and Ibuprofen treated groupcompare with EAE group (P<0.01). The transcriptional expression ofm-Calpain as examined by RT-PCR was not significantly increased in EAEgroup(P > 0.05). However, the expression of Calpain protein wassignificantly increased in EAE rats compare with normal control group(P<0.01). The efficacy between rosiglitazone and Ibuprofen was nosignificant different.
Conclusions: 1)The method of establishing the model of EAE wasstable and reliable in our study. 2)PPAR-γ agonists rosiglitazone andnon-steroidal antiinflammatory drugs (NSAIDs) Ibuprofen can improve theclinical symptoms and signs of EAE in rats, and down modulate the levelof the expression of TNF-αmRNA and Calpain protein. These resultssuggested that orally administered PPAR-γ agonists rosiglitazone andIbuprofen could provide therapeutic benefit in EAE or MS. 3)Our findingssuggested that Calpain played a major role in demyelination in EAE. Thelevel of the expression of Calpain protein indicates the efficacy of thetherapeutic drugs.
引文
[1] Paintlia AS, Paintlia MK, Singh AK, et al. Regulation of Gene Expression Associated With Acute Experimental Autoimmune Encephalomyelitis by Lovastatin [J]. Journal of Neuroscience Research 2004, 77: 63–81.
[2] Bertolotto A, Malucchi S , Capobianci M, et al. Quantitative PCR reveals increased level of tumor necrosis factor-alpha mRNA in peripheral blood mononuclear of multiple sclerosis patients during relapses[J]. J Interferon Cytokine Res,1999, 19(6):575-586.
[3] A ntella L, Kyozuka K, De Riso L, et al. Calcium, protease action, and the regulation of the cell cycle [J].Cell Calcium. 1998, 23(2-3):123-130.
[4] Schaecher KE, Shields DC, Banik NL. Mechanism of myelin breakdown iin experimental denyelination: a putative role for calpain [J]. Neurochemical research, 2001,26(6):731-737
[5] Shields DC, Tyor WR, DEIBLER GE, et al. Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis. [J] Proc. Natl. Acad. Sci. USA ,1998,95:5768–5772
[6] Mudaliar S and Henry RR. New oral therapies for type 2 diabetes mellitus: the glitazones or insulin sensitizers [J]. Annu Rev Med.2002,52:239-257
[7] Schmidt S, Moric E, Schmidt M, et al. Anti-inflammatory and antiproliferative actions of PPAR-γ agonists on T lymphocytes derived from MS patients [J]. Journal of Leukocyte Biology,2004,75:478-485
[8] Duvanel CB, Honegger P, Pershadsingh H, et al. Inhibition of Glial Cell Proinflammatory Activities by Peroxisome Proliferator-Activated Receptor Gamma Agonist Confers Partial Protection During Antimyelin Oligodendrocyte Glycoprotein Demyelination In Vitro [J]. Journal of Neuroscience Research, 2003,71:246–255
[9] Lehmann JM, Lenhard JM, Oliver BB, et al. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs [J]. J Biol Chem ,1997,272(6): 3406-10
[10]邢清和,郑荣远,王永铭,等,Wistar 大鼠过敏性脑脊髓炎动物模型的建立[J]。 上海医科大学学报,2000,27(6):471-473
[11]Koh CS, Paterson PY. Suppression of clinical signs of cell-transferred experimental allergic encephalomyelitis and altered cerebrovascular permeability in Lewis rats treated with a plasminogen activator inhibitor [J]. Cell-Immunol. 1987,107(1): 52-63
[12]董梅,刘瑞春,郭力,等,Wistar 大鼠实验性变态反应性脑脊髓炎的模型建立 [J]。 脑与神经疾病杂志,2004,12(6):425-429
[13]Feinstein DL, Galea E, Gavrilyuk V, et al. Peroxisome proliferator-activated receptor-γagonists prevent experimental autoimmune encephalomyelitis [J]. Ann Neurol 2002,51:694-702
[14]关洪平,李树新,库宝善,肿瘤坏死因子 α 与中枢神经系统 [J]。生理科学进展,1998,1(29):55-58
[15]Villarroya H, Violleau K, Ben-Younes-Chennoufi A, et al. Myelin-induced experimental allergic encephalomyelitis in Lewis rats: tumor necrosis factor alpha levels in serum and cerebrospinal fluid immunohistochemical expression in glial cells and macrophages of optic nerve and spinal cord [J]. J Neuroimmunol, 1996 ,64(1): 55-61
[16]Anonymous. TNF neutralization in MS: results of a randomized, placebo-controlled multicenter study. The Lenercept Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Analysis Group [J]. Neurology, 1999 ,53(3): 457-65
[17]Hohlfeld R, Meinl E, Weber F, et al. The role of autoimmune T lymphocytes in the pathogenesis of multiple sclerosis [J]. Neurology, 1995, 45(6 Suppl 6): S33-8.
[18]Diab A, Deng C, Smith JD, et al. Peroxisome proliferator-activated receptor-γ agonist 15-deoxy-?12, 14-prostaglandin J2 ameliorates experimental autoimmune encephalomyelitis[J].J Immunol,2002, 168:2508-2515.
[19]Niino M, Iwabuchi K, Kikuchi S, et al. Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by an agonist of peroxisome proliferatoractivated receptor-γ [J]. J Neuroimmunol, 2002, 116: 40–48.
[20]Neuhaus O, Archelos JJ, Hartung HP. Immunomodulation in multiple sclerosis: from immunosuppression to neuroprotection [J].Trends-Pharmacol-Sci. 2003,24(3):131-8
[21]Vamecq J, Latruffe N. Medical significance of peroxisome proliferatior-activated receptors [J]. LANCET, 1999, 354:141-148.
[22]Kielian T, Drew PD. Effects of Peroxisome Proliferator-Activated Receptor-γAgonists on Central Nervous System Inflammation [J]. Journal of Neuroscience Research,2003,71:315–325
[23]Adamson DJ, Frew D, Tatoud R, et al. Diclofenac antagonizes peroxisome proliferator-activated receptor γ signalling [J]. Mol Pharmacol, 2002, 61(1):7-12.
[24]Sastre M, Dewachter I, Landreth GE, et al. Nonsteroidal Anti-Inflammatory Drugs and Peroxisome Proliferator-Activated Receptor-γ Agonists Modulate Immunostimulated Processing of Amyloid Precursor Protein through Regulation of β-Secretase[J].The Journal of Neuroscience,2003 ,23(30):9796 –9804
[25]Fiebich BL, Hofer TJ, Lieb K, et al. The non-steroidal anti-inflammatory drug tepoxalin inhibits interleukin-6 and alphal-anti-chymotrypsin synthesis in astrocytes by preventing degradation of IkB-alpha [J]. Neuropharmacology, 1999,38:1325–133
[26]Nikitakis NG, Hebert C, Lopes MA, et al. PPARγ mediated antineoplastic effect of NSAID sulindac on human oral squamous carcinoma cells [J]. Int J Cancer,2002,98:817–23
[27]Hye-Kyung Na, Young-Joon. Surh Peroxisome proliferator-activated receptor gamma (PPARγ) ligands as bifunctional regulators of cell proliferation [J]. Biochemical Pharmacology, 2003, 66:1381–1391.
[28]Guroff GA. Neutral calcium-activated proteinase from the soluble fraction of rat [J]. Brain,1964, 239: 149-55
[29]Meyer WL, EH Fischer, EG Krebs. Activation of skeletal musclephosphorylase b by Ca2+ [J]. Biochemistry.1964,3:1033-39
[30]Shields DC, Schaecher KE, Goust JM, et al. Calpain activity and expression are increased in splenic inflammatory cells associated with experimental allergic encephalomyelitis [J]. J Neuroimmunol. 1999, 99(1):1-12.
[31]Antella L, Kyozuka K, De Riso L, et al. Calcium, protease action, and the regulation of the cell cycle .Cell Calcium [J].1998 ,23(2-3):123-30
[32]Ou BR, Forsberg NE. Determination of skeletal muscle Calpain and calpastatin activities during maturation [J]. Am J Physiol,1991,261(6 Pt 1):E677-83
[33]Shields DC, Schaecher KE, Saido TC, et al. A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, Calpain [J]. Proc Natl Acad Sci USA. 1999 ,96(20):11486-11491
[34]Yano T, Kobayashi A, Kurata S, et al. Purification and characterization of cathepsin B mRNA 3’-untranslated-regionbinding protein (CBBP), a protein that represses cathepsin B mRNA translation [J]. Eur J Biochem, 1997, 245:260-265.
[35]Schaecher. K, Rocchini A, Dinkins J, et al. Calpain expression and infiltrtiono factivated T cells in experimental allergic encephalomyelitisovtime: increased Calpain activity begins with on set of disease [J]. J Neuroimmunol, 2002, 129:1-9.
[36]Barnoy S, Kosower NS. Caspase-1-induced calpastatin degradation in myoblast differentiation and fusion: cross-talk between the caspase and Calpain systems [J]. FEBS Letters, 2003, 546:213-217.
[37]王国祥,王瑜,叶龙,等,钙激活中性蛋白酶抑制剂对实验性自身免疫性脑脊髓炎的保护作用 [J]。中国神经免疫学和神经病学杂志,2004,11(5),273-276
[2] Bertolotto A, Malucchi S , Capobianci M, et al. Quantitative PCR reveals increased level of tumor necrosis factor-alpha mRNA in peripheral blood mononuclear of multiple sclerosis patients during relapses[J]. J Interferon Cytokine Res,1999, 19(6):575-586.
[3] A ntella L, Kyozuka K, De Riso L, et al. Calcium, protease action, and the regulation of the cell cycle [J].Cell Calcium. 1998, 23(2-3):123-130.
[4] Schaecher KE, Shields DC, Banik NL. Mechanism of myelin breakdown iin experimental denyelination: a putative role for calpain [J]. Neurochemical research, 2001,26(6):731-737
[5] Shields DC, Tyor WR, DEIBLER GE, et al. Increased calpain expression in activated glial and inflammatory cells in experimental allergic encephalomyelitis. [J] Proc. Natl. Acad. Sci. USA ,1998,95:5768–5772
[6] Mudaliar S and Henry RR. New oral therapies for type 2 diabetes mellitus: the glitazones or insulin sensitizers [J]. Annu Rev Med.2002,52:239-257
[7] Schmidt S, Moric E, Schmidt M, et al. Anti-inflammatory and antiproliferative actions of PPAR-γ agonists on T lymphocytes derived from MS patients [J]. Journal of Leukocyte Biology,2004,75:478-485
[8] Duvanel CB, Honegger P, Pershadsingh H, et al. Inhibition of Glial Cell Proinflammatory Activities by Peroxisome Proliferator-Activated Receptor Gamma Agonist Confers Partial Protection During Antimyelin Oligodendrocyte Glycoprotein Demyelination In Vitro [J]. Journal of Neuroscience Research, 2003,71:246–255
[9] Lehmann JM, Lenhard JM, Oliver BB, et al. Peroxisome proliferator-activated receptors alpha and gamma are activated by indomethacin and other non-steroidal anti-inflammatory drugs [J]. J Biol Chem ,1997,272(6): 3406-10
[10]邢清和,郑荣远,王永铭,等,Wistar 大鼠过敏性脑脊髓炎动物模型的建立[J]。 上海医科大学学报,2000,27(6):471-473
[11]Koh CS, Paterson PY. Suppression of clinical signs of cell-transferred experimental allergic encephalomyelitis and altered cerebrovascular permeability in Lewis rats treated with a plasminogen activator inhibitor [J]. Cell-Immunol. 1987,107(1): 52-63
[12]董梅,刘瑞春,郭力,等,Wistar 大鼠实验性变态反应性脑脊髓炎的模型建立 [J]。 脑与神经疾病杂志,2004,12(6):425-429
[13]Feinstein DL, Galea E, Gavrilyuk V, et al. Peroxisome proliferator-activated receptor-γagonists prevent experimental autoimmune encephalomyelitis [J]. Ann Neurol 2002,51:694-702
[14]关洪平,李树新,库宝善,肿瘤坏死因子 α 与中枢神经系统 [J]。生理科学进展,1998,1(29):55-58
[15]Villarroya H, Violleau K, Ben-Younes-Chennoufi A, et al. Myelin-induced experimental allergic encephalomyelitis in Lewis rats: tumor necrosis factor alpha levels in serum and cerebrospinal fluid immunohistochemical expression in glial cells and macrophages of optic nerve and spinal cord [J]. J Neuroimmunol, 1996 ,64(1): 55-61
[16]Anonymous. TNF neutralization in MS: results of a randomized, placebo-controlled multicenter study. The Lenercept Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Analysis Group [J]. Neurology, 1999 ,53(3): 457-65
[17]Hohlfeld R, Meinl E, Weber F, et al. The role of autoimmune T lymphocytes in the pathogenesis of multiple sclerosis [J]. Neurology, 1995, 45(6 Suppl 6): S33-8.
[18]Diab A, Deng C, Smith JD, et al. Peroxisome proliferator-activated receptor-γ agonist 15-deoxy-?12, 14-prostaglandin J2 ameliorates experimental autoimmune encephalomyelitis[J].J Immunol,2002, 168:2508-2515.
[19]Niino M, Iwabuchi K, Kikuchi S, et al. Amelioration of experimental autoimmune encephalomyelitis in C57BL/6 mice by an agonist of peroxisome proliferatoractivated receptor-γ [J]. J Neuroimmunol, 2002, 116: 40–48.
[20]Neuhaus O, Archelos JJ, Hartung HP. Immunomodulation in multiple sclerosis: from immunosuppression to neuroprotection [J].Trends-Pharmacol-Sci. 2003,24(3):131-8
[21]Vamecq J, Latruffe N. Medical significance of peroxisome proliferatior-activated receptors [J]. LANCET, 1999, 354:141-148.
[22]Kielian T, Drew PD. Effects of Peroxisome Proliferator-Activated Receptor-γAgonists on Central Nervous System Inflammation [J]. Journal of Neuroscience Research,2003,71:315–325
[23]Adamson DJ, Frew D, Tatoud R, et al. Diclofenac antagonizes peroxisome proliferator-activated receptor γ signalling [J]. Mol Pharmacol, 2002, 61(1):7-12.
[24]Sastre M, Dewachter I, Landreth GE, et al. Nonsteroidal Anti-Inflammatory Drugs and Peroxisome Proliferator-Activated Receptor-γ Agonists Modulate Immunostimulated Processing of Amyloid Precursor Protein through Regulation of β-Secretase[J].The Journal of Neuroscience,2003 ,23(30):9796 –9804
[25]Fiebich BL, Hofer TJ, Lieb K, et al. The non-steroidal anti-inflammatory drug tepoxalin inhibits interleukin-6 and alphal-anti-chymotrypsin synthesis in astrocytes by preventing degradation of IkB-alpha [J]. Neuropharmacology, 1999,38:1325–133
[26]Nikitakis NG, Hebert C, Lopes MA, et al. PPARγ mediated antineoplastic effect of NSAID sulindac on human oral squamous carcinoma cells [J]. Int J Cancer,2002,98:817–23
[27]Hye-Kyung Na, Young-Joon. Surh Peroxisome proliferator-activated receptor gamma (PPARγ) ligands as bifunctional regulators of cell proliferation [J]. Biochemical Pharmacology, 2003, 66:1381–1391.
[28]Guroff GA. Neutral calcium-activated proteinase from the soluble fraction of rat [J]. Brain,1964, 239: 149-55
[29]Meyer WL, EH Fischer, EG Krebs. Activation of skeletal musclephosphorylase b by Ca2+ [J]. Biochemistry.1964,3:1033-39
[30]Shields DC, Schaecher KE, Goust JM, et al. Calpain activity and expression are increased in splenic inflammatory cells associated with experimental allergic encephalomyelitis [J]. J Neuroimmunol. 1999, 99(1):1-12.
[31]Antella L, Kyozuka K, De Riso L, et al. Calcium, protease action, and the regulation of the cell cycle .Cell Calcium [J].1998 ,23(2-3):123-30
[32]Ou BR, Forsberg NE. Determination of skeletal muscle Calpain and calpastatin activities during maturation [J]. Am J Physiol,1991,261(6 Pt 1):E677-83
[33]Shields DC, Schaecher KE, Saido TC, et al. A putative mechanism of demyelination in multiple sclerosis by a proteolytic enzyme, Calpain [J]. Proc Natl Acad Sci USA. 1999 ,96(20):11486-11491
[34]Yano T, Kobayashi A, Kurata S, et al. Purification and characterization of cathepsin B mRNA 3’-untranslated-regionbinding protein (CBBP), a protein that represses cathepsin B mRNA translation [J]. Eur J Biochem, 1997, 245:260-265.
[35]Schaecher. K, Rocchini A, Dinkins J, et al. Calpain expression and infiltrtiono factivated T cells in experimental allergic encephalomyelitisovtime: increased Calpain activity begins with on set of disease [J]. J Neuroimmunol, 2002, 129:1-9.
[36]Barnoy S, Kosower NS. Caspase-1-induced calpastatin degradation in myoblast differentiation and fusion: cross-talk between the caspase and Calpain systems [J]. FEBS Letters, 2003, 546:213-217.
[37]王国祥,王瑜,叶龙,等,钙激活中性蛋白酶抑制剂对实验性自身免疫性脑脊髓炎的保护作用 [J]。中国神经免疫学和神经病学杂志,2004,11(5),273-276