胸腺五肽治疗实验性自身免疫性脑脊髓炎大鼠的实验研究
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摘要
目的:
多发性硬化(multiple sclerosis,MS)是中枢神经系统(central nervous system,CNS)白质脱髓鞘病变为特点的自身免疫性疾病。具有反复发作,部分患者病情呈进行性加重的特点。由于MS发病率较高,呈慢性病程且倾向于青年人罹患,使其成为重要的神经系统疾病之一。MS发病机制不确切,尚缺乏行之有效的防治措施,因此通过建立动物模型,探讨MS的发病机制,寻求有效的治疗方案,是我们目前研究的主要方向。
胸腺五肽(Thymopoietin,TP-5)是从胸腺激素中分离出来的胸腺生成素Ⅱ功能活性部分,胸腺生成素Ⅱ由49个氨基酸组成,而其中由5个氨基酸组成的肽链片段,有着与胸腺生成素Ⅱ相同的全部生理功能,所以就把这个五肽片段称为胸腺五肽。在实验研究和临床应用中,胸腺五肽均显示出具有调节免疫系统的作用,能诱导T淋巴细胞及其亚群分化和成熟;减少自身抗体的产生;调节T淋巴亚群比例,使CD4~+/CD8~+趋于正常,免疫系统恢复正常。在某些自身免疫性疾病(如类风湿性关节炎,系统性红斑狼疮等)中疗效明显,长期应用耐受良好。但目前国内外应用胸腺五肽治疗EAE/MS尚未见报道。
本实验旨在MS的经典动物模型—实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)基础上,探讨不同剂量的胸腺五肽对EAE的治疗作用及其机制,为脱髓鞘疾病的研究提供新的思路。
方法:
1动物分组:将78只6～8周龄雌性Wistar大鼠,体重180～200g,随机分为正常对照组6只,EAE模型组、地塞米松(dexamethasone,DXM)组、TP-5小剂量组(0.05 mg·kg~(-1)·d~(-1))、TP-5大剂量组(0.25 mg·kg~(-1)·d~(-1))各18只。每组又随机分为7天、14天、21天三个亚组,每亚组6只大鼠。
2模型制备:将新鲜豚鼠全脊髓制成匀浆,加上完全福氏佐剂(complete Freund's adjuvant,CFA),佐剂中含卡介苗4 mg/mL,经过乳化后经按0.4 mL/只分别于大鼠四肢足垫皮下注射。药物治疗组于模型制备第一天起给予相应剂量腹腔注射,药物每天一次持续给至动物处死时为止。
3神经功能评分:于免疫诱导当日及免疫后每天对动物的体重变化、精神状态、活动情况进行观察,并对动物进行神经功能评分,采用Kono等提出的标准,分为5分:1分动物尾部无力;2分尾部无力+肢体无力;3分肢体轻度麻痹;4分级肢体严重麻痹,被动翻身后不能复原;5分濒死状态。
4组织病理学:分别于免疫后第7天、14天、21天,在动物麻醉状态下,快速取其大脑侧脑室周围及颈、胸、腰膨大处组织,40g/L多聚甲醛固定,石蜡包埋切片,行HE染色,光学纤维镜下观察炎症细胞浸润情况。
5 RT-PCR法检测脑组织IFN-γ、IL-4 mRNA的表达:实验动物于免疫后第7天、14天、21天,在麻醉状态下,快速取大脑侧脑室周围及颈、胸、腰膨大处组织,液氮中保存,Trizol一步法提取CNS组织总RNA,采用随机引物法进行逆转录反应后,扩增获得目的基因IFN-γ、IL-4 RT-PCR产物,以β-actin作为内对照。最后紫外凝胶成像分析系统分析结果。
结果:
1动物发病率观察:TP-5小剂量组、DXM组低于EAE组及TP-5大剂量组,均有统计学意义(P＜0.01)。DXM组低于TP-5小剂量组,有统计学意义(P＜0.01)。EAE组与TP-5大剂量组之间的发病率没有统计学意义(P＞0.05)。
2行为学观察
2.1大鼠在发病潜伏期和缓解期时神经功能评分比较:各组之间无明显差别。
2.2各组大鼠在发病高峰期时神经功能评分比较:TP-5小剂量组、DXM组各大鼠神经功能评分明显低于EAE组及TP-5大剂量组(P＜0.01)。DXM组明显低于TP-5小剂量组(P＜0.01)。EAE组与TP-5大剂量组之间神经功能评分无明显差别(P＞0.05)。
3组织病理学观察:在疾病早期,EAE鼠组织内即出现炎性细胞浸润,且炎性细胞数目的多少与疾病严重程度相一致。在发病高峰期和恢复期,TP-5小剂量组、DXM组炎症细胞浸润形成的血管套袖数目明显低于EAE组及TP-5大剂量组(P＜0.01)。DXM组和TP-5小剂量组,EAE组和TP-5大剂量组炎症细胞浸润形成的血管套袖数目没有统计学意义(P＞0.05)。
4 IFN-γmRNA、IL-4 mRNA表达
4.1 IFN-γmRNA:除正常对照组外,其余各组IFN-γmRNA在潜伏期即表达上调,随着进入症状高峰期,IFN-γmRNA表达水平亦达高峰,恢复期IFN-γmRNA表达水平下降。在发病高峰期和恢复期,DXM组和TP-5小剂量组IFN-γmRNA表达水平与EAE组及TP-5大剂量组相比明显下降(P＜0.01)。DXM组与TP-5小剂量组间,EAE组和TP-5大剂量组之间比较差异无统计学意义(P＞0.05)。
4.2 IL-4 mRNA:IL-4 mRNA表达水平呈逐渐升高的趋势,潜伏期和症状高峰期IL-4 mRNA有所表达,恢复期达峰值。在发病高峰期和恢复期,DXM组和TP-5小剂量组IL-4mRNA表达明显高于EAE组(P＜0.01),DXM组与TP-5小剂量组间比较差异无统计学意义(P＞0.05)。
结论:
1胸腺五肽可以降低EAE大鼠的发病率,减少疾病对机体的神经功能损害。
2胸腺五肽可以减少EAE大鼠脑组织IFN-γmRNA的表达,减少中枢神经系统内炎症细胞浸润的程度。
3胸腺五肽可以提高EAE大鼠脑组织IL-4 mRNA表达,促进病情缓解。
4胸腺五肽对MS的治疗作用不呈剂量依赖性。
5胸腺五肽与地塞米松比较,虽然地塞米松在发病率和神经功能评分上优于胸腺五肽,但是由于胸腺五肽可以避免长期应用地塞米松引起的不良反应,充分显示出应用安全和耐受性良好的优势。
Objective
Multiple sclerosis(MS)is an inflammatory demyelination disease of the central nervous system(CNS)that causes relapsing and progressive neurological impairment.The disease-modifying agents currently used in the treatment of MS are not completely effective.It is our main aim that we investigate the pathogenesis of MS and seek effective therapeutic scheme for MS by setting up an animal model.
Thymopentin(TP-5)is a synthetic pentapeptide corresponding to amino acids 32-36 of the thymic polypeptide thymopoietinⅡ,which contains 49 amino acids.TP-5 has the whole physiological function of thymopoietinⅡ.TP-5,as an immunomodulator,has been shown to induce T lymphocyte differentiation and activation,reduce the production of the autoantibodies,normalize the level of CD4~+/CD8~+ and regulate the immune reconstitution.The effect of TP-5 is significantly in some autoimmune diseases such as rheumatoid arthritis(RA) and systemic lupus erythematosus(SLE).
In our study,we administrate TP-5 to the rats of experimental autoimmune encephalomyelitis(EAE)-an animal model of MS,in order to investigate the therapeutic potential in treatment of neuroinflammatory diseases like MS.
Methods
Seventy-eight healthy female Wistar rats weighing 180～200g were divided randomly into five groups:normal control group(6 rats),EAE group(18 rats),dexamethasone(DXM) group(18 rats),low dose of TP-5 group(18 rats)and high dose of TP-5 group(18 rats).EAE group and treatment groups were divided three groups:7 day group,14 day group,21 day group, separately.All experimental rats were immunized subcutaneously in the Wistar rats four foot pads by fresh guinea pig spinal cord homogenate(GPSCH)and complete Freund's adjuvant(CFA).Clinical signs of EAE were assessed the mean of twice daily by two observations.Scores were assigned on the basis of the following symptoms:1,tail weakness;2,tail weakness plus limb asthenia;3,mild limb paralysis;4,severe limb paralysis;5,moribund/dead.EAE group,DXM group,low dose of TP-5 group and high dose of TP-5 group were injected intraperitoneal respectively NS 0.5mL/d、DXM 5 mg·kg~(-1)·d~(-1)、TP-5 0.05 mg·kg~(-1)·d~(-1)、TP-5 0.25mg·kg~(-1)·d~(-1).This treatment was started on the first day of immunization and continued daily for the duration of the experiment.During the experiment,the mean maximal score of animals at different time point and the incidence of disease were observed as results.
Rats were sacrificed after anesthesia with intraperitoneal injection.Tissues of the brain and spinal cord were fixed with 4 %formalin,then the tissues were embedded in paraffin and sectioned at 4μm thickness.Some of the sections were stained with HE to assess lymphocyte infiltration and inflammation.
The expression of cytokines IFN-γand IL-4 mRNA in the brain of rats was measured by semiquantitative reverse-transcription polymerase chain reaction(RT-PCR)on days of 7,14 and 21 post immunization(p.i.).
Results
1 The morbidity of disease in different group
Morbidity of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).
2 Clinical profile of EAE in different group
Neurological deficits scores of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).
3 Neuropathological findings
The results demonstrated that some monocytes infiltration was observed in the tissues of brains and spinal cords before the clinical signs emerging.The degree of infiltration was associated with the severity of EAE.The extent of inflammation of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).
4 Expression of IFN-γand IL-4 mRNA
In low dose of TP-5 group,the expression of IFN-γmRNA significantly decreased(P＜0.01)and that of IL-4 mRNA significantly increased on days of 14 and 21 post immunization (P＜0.01).
Conclusion
1 TP-5 reduces the morbidity,and protects the rats from the severity of the disease.
2 TP-5 can reduce the expression of IFN-γmRNA,and then reduces the lymphocyte infiltration and inflammation in the CNS of EAE rats.
3 TP-5 can increase the expression of IL-4 mRNA,which is associated recovery of EAE rats.
4 The effect of TP-5 on the EAE is not dose-dependent.
5 TP-5 has good safty,and tolerance compared with DXM. These results suggest that TP-5 may be a useful immunomodulator for demyelinating diseases without suppressing immune response.
多发性硬化(multiple sclerosis,MS)是中枢神经系统(central nervous system,CNS)白质脱髓鞘病变为特点的自身免疫性疾病。具有反复发作,部分患者病情呈进行性加重的特点。由于MS发病率较高,呈慢性病程且倾向于青年人罹患,使其成为重要的神经系统疾病之一。MS发病机制不确切,尚缺乏行之有效的防治措施,因此通过建立动物模型,探讨MS的发病机制,寻求有效的治疗方案,是我们目前研究的主要方向。
胸腺五肽(Thymopoietin,TP-5)是从胸腺激素中分离出来的胸腺生成素Ⅱ功能活性部分,胸腺生成素Ⅱ由49个氨基酸组成,而其中由5个氨基酸组成的肽链片段,有着与胸腺生成素Ⅱ相同的全部生理功能,所以就把这个五肽片段称为胸腺五肽。在实验研究和临床应用中,胸腺五肽均显示出具有调节免疫系统的作用,能诱导T淋巴细胞及其亚群分化和成熟;减少自身抗体的产生;调节T淋巴亚群比例,使CD4~+/CD8~+趋于正常,免疫系统恢复正常。在某些自身免疫性疾病(如类风湿性关节炎,系统性红斑狼疮等)中疗效明显,长期应用耐受良好。但目前国内外应用胸腺五肽治疗EAE/MS尚未见报道。
本实验旨在MS的经典动物模型—实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)基础上,探讨不同剂量的胸腺五肽对EAE的治疗作用及其机制,为脱髓鞘疾病的研究提供新的思路。
方法:
1动物分组:将78只6～8周龄雌性Wistar大鼠,体重180～200g,随机分为正常对照组6只,EAE模型组、地塞米松(dexamethasone,DXM)组、TP-5小剂量组(0.05 mg·kg~(-1)·d~(-1))、TP-5大剂量组(0.25 mg·kg~(-1)·d~(-1))各18只。每组又随机分为7天、14天、21天三个亚组,每亚组6只大鼠。
2模型制备:将新鲜豚鼠全脊髓制成匀浆,加上完全福氏佐剂(complete Freund's adjuvant,CFA),佐剂中含卡介苗4 mg/mL,经过乳化后经按0.4 mL/只分别于大鼠四肢足垫皮下注射。药物治疗组于模型制备第一天起给予相应剂量腹腔注射,药物每天一次持续给至动物处死时为止。
3神经功能评分:于免疫诱导当日及免疫后每天对动物的体重变化、精神状态、活动情况进行观察,并对动物进行神经功能评分,采用Kono等提出的标准,分为5分:1分动物尾部无力;2分尾部无力+肢体无力;3分肢体轻度麻痹;4分级肢体严重麻痹,被动翻身后不能复原;5分濒死状态。
4组织病理学:分别于免疫后第7天、14天、21天,在动物麻醉状态下,快速取其大脑侧脑室周围及颈、胸、腰膨大处组织,40g/L多聚甲醛固定,石蜡包埋切片,行HE染色,光学纤维镜下观察炎症细胞浸润情况。
5 RT-PCR法检测脑组织IFN-γ、IL-4 mRNA的表达:实验动物于免疫后第7天、14天、21天,在麻醉状态下,快速取大脑侧脑室周围及颈、胸、腰膨大处组织,液氮中保存,Trizol一步法提取CNS组织总RNA,采用随机引物法进行逆转录反应后,扩增获得目的基因IFN-γ、IL-4 RT-PCR产物,以β-actin作为内对照。最后紫外凝胶成像分析系统分析结果。
结果:
1动物发病率观察:TP-5小剂量组、DXM组低于EAE组及TP-5大剂量组,均有统计学意义(P＜0.01)。DXM组低于TP-5小剂量组,有统计学意义(P＜0.01)。EAE组与TP-5大剂量组之间的发病率没有统计学意义(P＞0.05)。
2行为学观察
2.1大鼠在发病潜伏期和缓解期时神经功能评分比较:各组之间无明显差别。
2.2各组大鼠在发病高峰期时神经功能评分比较:TP-5小剂量组、DXM组各大鼠神经功能评分明显低于EAE组及TP-5大剂量组(P＜0.01)。DXM组明显低于TP-5小剂量组(P＜0.01)。EAE组与TP-5大剂量组之间神经功能评分无明显差别(P＞0.05)。
3组织病理学观察:在疾病早期,EAE鼠组织内即出现炎性细胞浸润,且炎性细胞数目的多少与疾病严重程度相一致。在发病高峰期和恢复期,TP-5小剂量组、DXM组炎症细胞浸润形成的血管套袖数目明显低于EAE组及TP-5大剂量组(P＜0.01)。DXM组和TP-5小剂量组,EAE组和TP-5大剂量组炎症细胞浸润形成的血管套袖数目没有统计学意义(P＞0.05)。
4 IFN-γmRNA、IL-4 mRNA表达
4.1 IFN-γmRNA:除正常对照组外,其余各组IFN-γmRNA在潜伏期即表达上调,随着进入症状高峰期,IFN-γmRNA表达水平亦达高峰,恢复期IFN-γmRNA表达水平下降。在发病高峰期和恢复期,DXM组和TP-5小剂量组IFN-γmRNA表达水平与EAE组及TP-5大剂量组相比明显下降(P＜0.01)。DXM组与TP-5小剂量组间,EAE组和TP-5大剂量组之间比较差异无统计学意义(P＞0.05)。
4.2 IL-4 mRNA:IL-4 mRNA表达水平呈逐渐升高的趋势,潜伏期和症状高峰期IL-4 mRNA有所表达,恢复期达峰值。在发病高峰期和恢复期,DXM组和TP-5小剂量组IL-4mRNA表达明显高于EAE组(P＜0.01),DXM组与TP-5小剂量组间比较差异无统计学意义(P＞0.05)。
结论:
1胸腺五肽可以降低EAE大鼠的发病率,减少疾病对机体的神经功能损害。
2胸腺五肽可以减少EAE大鼠脑组织IFN-γmRNA的表达,减少中枢神经系统内炎症细胞浸润的程度。
3胸腺五肽可以提高EAE大鼠脑组织IL-4 mRNA表达,促进病情缓解。
4胸腺五肽对MS的治疗作用不呈剂量依赖性。
5胸腺五肽与地塞米松比较,虽然地塞米松在发病率和神经功能评分上优于胸腺五肽,但是由于胸腺五肽可以避免长期应用地塞米松引起的不良反应,充分显示出应用安全和耐受性良好的优势。
Objective
Multiple sclerosis(MS)is an inflammatory demyelination disease of the central nervous system(CNS)that causes relapsing and progressive neurological impairment.The disease-modifying agents currently used in the treatment of MS are not completely effective.It is our main aim that we investigate the pathogenesis of MS and seek effective therapeutic scheme for MS by setting up an animal model.
Thymopentin(TP-5)is a synthetic pentapeptide corresponding to amino acids 32-36 of the thymic polypeptide thymopoietinⅡ,which contains 49 amino acids.TP-5 has the whole physiological function of thymopoietinⅡ.TP-5,as an immunomodulator,has been shown to induce T lymphocyte differentiation and activation,reduce the production of the autoantibodies,normalize the level of CD4~+/CD8~+ and regulate the immune reconstitution.The effect of TP-5 is significantly in some autoimmune diseases such as rheumatoid arthritis(RA) and systemic lupus erythematosus(SLE).
In our study,we administrate TP-5 to the rats of experimental autoimmune encephalomyelitis(EAE)-an animal model of MS,in order to investigate the therapeutic potential in treatment of neuroinflammatory diseases like MS.
Methods
Seventy-eight healthy female Wistar rats weighing 180～200g were divided randomly into five groups:normal control group(6 rats),EAE group(18 rats),dexamethasone(DXM) group(18 rats),low dose of TP-5 group(18 rats)and high dose of TP-5 group(18 rats).EAE group and treatment groups were divided three groups:7 day group,14 day group,21 day group, separately.All experimental rats were immunized subcutaneously in the Wistar rats four foot pads by fresh guinea pig spinal cord homogenate(GPSCH)and complete Freund's adjuvant(CFA).Clinical signs of EAE were assessed the mean of twice daily by two observations.Scores were assigned on the basis of the following symptoms:1,tail weakness;2,tail weakness plus limb asthenia;3,mild limb paralysis;4,severe limb paralysis;5,moribund/dead.EAE group,DXM group,low dose of TP-5 group and high dose of TP-5 group were injected intraperitoneal respectively NS 0.5mL/d、DXM 5 mg·kg~(-1)·d~(-1)、TP-5 0.05 mg·kg~(-1)·d~(-1)、TP-5 0.25mg·kg~(-1)·d~(-1).This treatment was started on the first day of immunization and continued daily for the duration of the experiment.During the experiment,the mean maximal score of animals at different time point and the incidence of disease were observed as results.
Rats were sacrificed after anesthesia with intraperitoneal injection.Tissues of the brain and spinal cord were fixed with 4 %formalin,then the tissues were embedded in paraffin and sectioned at 4μm thickness.Some of the sections were stained with HE to assess lymphocyte infiltration and inflammation.
The expression of cytokines IFN-γand IL-4 mRNA in the brain of rats was measured by semiquantitative reverse-transcription polymerase chain reaction(RT-PCR)on days of 7,14 and 21 post immunization(p.i.).
Results
1 The morbidity of disease in different group
Morbidity of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).
2 Clinical profile of EAE in different group
Neurological deficits scores of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).
3 Neuropathological findings
The results demonstrated that some monocytes infiltration was observed in the tissues of brains and spinal cords before the clinical signs emerging.The degree of infiltration was associated with the severity of EAE.The extent of inflammation of low dose of TP-5 group and DXM group were significantly lower than those of EAE group and high dose of TP-5 group(P＜0.01).
4 Expression of IFN-γand IL-4 mRNA
In low dose of TP-5 group,the expression of IFN-γmRNA significantly decreased(P＜0.01)and that of IL-4 mRNA significantly increased on days of 14 and 21 post immunization (P＜0.01).
Conclusion
1 TP-5 reduces the morbidity,and protects the rats from the severity of the disease.
2 TP-5 can reduce the expression of IFN-γmRNA,and then reduces the lymphocyte infiltration and inflammation in the CNS of EAE rats.
3 TP-5 can increase the expression of IL-4 mRNA,which is associated recovery of EAE rats.
4 The effect of TP-5 on the EAE is not dose-dependent.
5 TP-5 has good safty,and tolerance compared with DXM. These results suggest that TP-5 may be a useful immunomodulator for demyelinating diseases without suppressing immune response.
引文
1 Karpus WJ, Ransohoff RM. Chemokine regulation of experimental autoimmune encephalomyelitis: temporal and spatial expression patterns govern disease pathogenesis. J Immunol, 1998,161(6): 2667-2671
2 Mancardi G, Hart BA, Capello E, et al. Restricted immune responses lead to CNS demyelination and axonal damage. J Neuroimmunol, 2000,107(2): 178-183
3 Sundal E, Bertelletti D. Thymopentin treatment of rheumatoid arthritis. Arzneimittelforschung, 1994, 44(10): 1145-1149
4 Kono DH, Urban JL, Horvath SJ, et al. Two minor determinants of myelin basic protein induce experimental allergic encephalomyelitis in SJL/J mice. J Exp Med, 1988, 168(1): 213-227
5 McCombe PA, Nickson I, Pender MP. Cytokine expression by inflammatory cells obtained from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis induced by inoculation with myelin basic protein and adjuvants. J Neuroimmunol, 1998(1-2):30-38
6 Graciela L, Boccaccio, Felix Mor, et al. Non-coding plasmid DNA induces IFY-γ in vivo and suppresses autoimmune encephalomyelitis. Int Immunol, 1999, 11 (2): 289-296
7 Clanet M, Radue EW, Kappos L, et al. A randomized, double-blind,dose-comparison study of weekly interferon beta-la in relapsing MS.Neurology,2002,59(10):1507-1517
8 Sipe JC,Romine JS,Koziol JA,et al.Cladribine in treatment of chronic progressive multiple sclerosis.Lancet,1994,344(8914):9-13
9 Currier RD,Haerer AF,Meydrech EF.Low dose oral methotrexate treatment of multiple sclerosis:a pilot study.J Neurol Neurosurg Psychiatry,1993,56(11):1217-1218
10 王勤,杨金波,胡晓愚.胸腺生成素Ⅱ活性片段TP-5增强免疫功能的剂量效应.兰州大学学报,1992,28(3):128-32
11 张符光,刘佃辛.胸腺和胸腺素研究进展.《国外医学》免疫学分册,1996,4,187-190
12 蒋定文,李楚芳.胸腺五肽研究进展.《国外医学》预防、诊断、治疗用生物制品分册,1999,22(2):69-72
13 Rivero VE,Maccioni M,Bucher AE,et al.Suppression of experimental autoimmune encephalomyelitis(EAE)by intraperitoneal administration of soluble myelin antigens in Wistar rats.J Neuroimmunol,1997,72(1):3-10
14 Yu CY,Whitacre CC.Sex,MHC and complement C4 in autoimmune diseases.Trends Immunol,2004,25(12):694-699
15 Maatta JA,Eralinna JP,Roytta M,et al.Physical state of the neuroantigen in adjuvant emulsions determines encephalitogenic status in the BALB/c mouse.J Immunol Method,1996,190(1):133-141
16 刑清和,王永铭,郑荣远.影响EAE动物模型建立的因素分析.中国临床神经科学,2000,8(4):305
17 Goverman J,Perchellet A,Huseby ES.The role of CD8(+)T cells in multiple sclerosis and its animal models.Curr Drug Targets Inflamm Allergy,2005,4(2):239-245
18 Zakrzewska KE,Cusin I,Stricker-Krongrad A,et al.Induction of obesity and hyperleptinemia by central glucocorticoid infusion in the rat.Diabetes,1999,48(2):365-370
19 Imitola J,Chitnis T,Khoury SJ.Cytokines in multiple sclerosis:from bench to bedside.Pharmacol Ther,2005,106(2):163-177
20 Abbas AK,Murphy KM,Sher A.Functional diversity of helper T lymphocytes.Nature,1996,383(6603):787-793.
21 Barten DM,Ruddle NH.Vascular cell adhesion molecule-1modulation by tumor necrosis factor in experimental allergic encephalomyelitis.J Neuroimmunol,1994,51(2):123-133
22 Issazadeh S,Mustafa M,Ljungdahl A,et al.Interferon gamma,interleukin 4 and transforming growth factor beta in experimental autoimmune encephalomyelitis in Lewis rats:dynamics of cellular mRNA expression in the central nervous system and lymphoid cells.J Neurosci Res,1995,40(5):579-590
23 Issazadeh S, Navikas V, Schaub M, et al. Kinetics of expression of costimulatory molecules and their ligands in murine relapsing experimental autoimmune encephalomyelitis in vivo. J Immunol, 1998, 161(3): 1104-1112
24 Begolka WS, Vanderlugt CL, Rahbe SM, et al.Differential expression of inflammatory cytokines parallels progression of central nervous system pathology in two clinically distinct models of multiple sclerosis. J Immunol, 1998, 161(8): 4437-4446
25 Chitnis T, Khoury SJ.Cytokine shifts and tolerance in experimental autoimmune encephalomyelitis. Immunol Res, 2003,28(3):223-239
26 Inobe J, Slavin AJ, Komagata Y, et al. IL-4 is a differenttiation factor for transforming growth factor-beta secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis. Eur J Immunol, 1998,28(9): 2780-2790
27 Kuroda Y, Shimamoto Y. Human tumor necrosis factor-alpha augments experimental allergic encephalomyelitis in rats.J Neuroimmunol, 1991, 34(2-3): 159-164
28 Lauritsen JP, Christensen MD, Dietrich J, et al. Two distinct pathways exist for down-regulation of the TCR. J Immunol. 1998,161(1):260-267
29 Sela M. Immunomodulatory vaccines against autoimmune diseases.Rejuvenation Res, 2006, 9(1): 126-133
30 Butti E, Bergami A, Recchia A, et al. IL4 gene delivery to the CNS recruits regulatory T cells and induces clinical recovery in mouse models of multiple sclerosis.Gene Ther. 2008,15(7):504-515
1 Noseworthy JH,Lucchinetti C,Rodriguez M,et al Multiple Sclerosis[J].N Engl J Med,2000,343(13):938-952
2 莫晔,刘胜利.多发性硬化症20例临床分析[J].临床荟萃,1997,13(6):274-275
3 Hartung HP,Gonsette R,Konig N,et al.Mitoxantrone in progressive multiple sclerosis:a placebo-controlled,doubleblind,randomized,multicentre trial[J].Lancet,2002,360(9350):2018-2025
4 Patti F,Amato MP,Filippi M,et al.A double blind,placebocontrolled,phase Ⅱ,add-on study of cyclophosphamide(CTX)for 24 months in patients affected by multiple sclerosis on a background therapy with interferon-beta study denomination:CYCLIN[J].J Neurol Sci,2004,223(1):69-71
5 Achiron A,Kishner I,Sarova-Pinhas I,et al.Intravenous immunoglobulin treatment following the first demyelinating event suggestive of multiple sclerosis:a randomized,double-blind,placebo-controlled trial[J].Arch Neurol,2004,61(10):1515-1520
6 Blanco Y,Moral EA,Costa M,et al.Effect of glatiramer acetate(Copaxone) on the immuno- phenotypic and cytokine profile and BDNF production in multiple sclerosis:a longitudinal study [J].Neurosci Lett,2006,406(3):270-275
7 Martinelli-Boneschi F, Rovaris M, Johnson KP,et al.Effects of glatiramer acetate on relapse rate and accumulated disability in multiple sclerosisrmeta-analysis of three double-blind,randomized,placebo-controlled clinical trials [J]. Multiple Sclerosis,2003,9(4):349-355
8 Rubio-Terres C,Aristegui Ruiz I, Medina Redondo F,et al.Cost-utility analisys of multiple sclerosis treatment with glatiramer acetate or interferon beta in Spain[J]. Farm Hosp, 2003,27(3):159-165
9 Clanet M,Radue EW,Kappos L,et al.A randomized, double- blind,dose-comparison study of weekly interferon beta-1a in relapsing MS[J].Neurology,2002,59(10): 1507-1517
10 Coppola G,Lanzillo R,Florio C,et al.Long-term clinical experience with weekly interferon beta-1a in relapsing multiple sclerosis[J].Eur J Neurol,2006,13(9): 1014-1021
11 PRISMS(Prevention of Relapses and Disability by interferon beta-1a Subcutaneously in Multiple Sclerosis) Study Group and the University of British Columbia MS/MRI Analysis Group. PRISMS-4: Long-term efficacy of interferon-beta-1a in relapsing MS[J] .Neurology, 2001,56(12):1628-1636
12 European Study Group on interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon beta-1b in treatment of secondary progressive multiple sclerosis[J].Lancet, 1998,352(9139): 1491-1497
13 Vollmer T, Key L, Durkalski V, et al. Oral simvastatin treatment in relapsing-remitting multiple sclerosis[J].Lancet, 2004,363(9421): 1607-1608
14 Stuve O,Youssef S,Steinman L,et al. Statins as potential therapeutic agents in neuro-inflammatory disorders[J].Curr Opin Neurol,2003,16(3):393-401
15 Bielekova B,Catalfamo M,Reichert-Scrivner S,et al. Regulatory CD56(bright) natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis[J].Proc Natl Acad Sci USA,2006,103(15):5941-5946
16 Pluchino S,Quattrini A,Brambilla E,et al.Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis[J].Nature,2003,422(6933):688-694
17 Achiron A,Mandel M. T-cell vaccination in multiple sclerosis[J].Autoimmun Rev,2004,3(1): 25-32
18 Direskeneli H,Eksioglu-Demiralp E,Kibaroglu A,et al. Oligoclonal T cell expansions in patients with Behcet's disease[J].Clin Exp Immunol, 1999,117(1):166-170
19 Popovic N,Schubart A,Goetz BD,et al.Inhibition of autoimmune encephalomyelitis by a tetracycline[J].Ann Neurol, 2002,51(2):215-223
20 Metz LM,Zhang Y,Yeung M,et al.Minocycline reduces gadolinium-enhancing magnetic resonance imaging lesions in multiple sclerosis[J].Ann Neurol,2004,55(5):756
2 Mancardi G, Hart BA, Capello E, et al. Restricted immune responses lead to CNS demyelination and axonal damage. J Neuroimmunol, 2000,107(2): 178-183
3 Sundal E, Bertelletti D. Thymopentin treatment of rheumatoid arthritis. Arzneimittelforschung, 1994, 44(10): 1145-1149
4 Kono DH, Urban JL, Horvath SJ, et al. Two minor determinants of myelin basic protein induce experimental allergic encephalomyelitis in SJL/J mice. J Exp Med, 1988, 168(1): 213-227
5 McCombe PA, Nickson I, Pender MP. Cytokine expression by inflammatory cells obtained from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis induced by inoculation with myelin basic protein and adjuvants. J Neuroimmunol, 1998(1-2):30-38
6 Graciela L, Boccaccio, Felix Mor, et al. Non-coding plasmid DNA induces IFY-γ in vivo and suppresses autoimmune encephalomyelitis. Int Immunol, 1999, 11 (2): 289-296
7 Clanet M, Radue EW, Kappos L, et al. A randomized, double-blind,dose-comparison study of weekly interferon beta-la in relapsing MS.Neurology,2002,59(10):1507-1517
8 Sipe JC,Romine JS,Koziol JA,et al.Cladribine in treatment of chronic progressive multiple sclerosis.Lancet,1994,344(8914):9-13
9 Currier RD,Haerer AF,Meydrech EF.Low dose oral methotrexate treatment of multiple sclerosis:a pilot study.J Neurol Neurosurg Psychiatry,1993,56(11):1217-1218
10 王勤,杨金波,胡晓愚.胸腺生成素Ⅱ活性片段TP-5增强免疫功能的剂量效应.兰州大学学报,1992,28(3):128-32
11 张符光,刘佃辛.胸腺和胸腺素研究进展.《国外医学》免疫学分册,1996,4,187-190
12 蒋定文,李楚芳.胸腺五肽研究进展.《国外医学》预防、诊断、治疗用生物制品分册,1999,22(2):69-72
13 Rivero VE,Maccioni M,Bucher AE,et al.Suppression of experimental autoimmune encephalomyelitis(EAE)by intraperitoneal administration of soluble myelin antigens in Wistar rats.J Neuroimmunol,1997,72(1):3-10
14 Yu CY,Whitacre CC.Sex,MHC and complement C4 in autoimmune diseases.Trends Immunol,2004,25(12):694-699
15 Maatta JA,Eralinna JP,Roytta M,et al.Physical state of the neuroantigen in adjuvant emulsions determines encephalitogenic status in the BALB/c mouse.J Immunol Method,1996,190(1):133-141
16 刑清和,王永铭,郑荣远.影响EAE动物模型建立的因素分析.中国临床神经科学,2000,8(4):305
17 Goverman J,Perchellet A,Huseby ES.The role of CD8(+)T cells in multiple sclerosis and its animal models.Curr Drug Targets Inflamm Allergy,2005,4(2):239-245
18 Zakrzewska KE,Cusin I,Stricker-Krongrad A,et al.Induction of obesity and hyperleptinemia by central glucocorticoid infusion in the rat.Diabetes,1999,48(2):365-370
19 Imitola J,Chitnis T,Khoury SJ.Cytokines in multiple sclerosis:from bench to bedside.Pharmacol Ther,2005,106(2):163-177
20 Abbas AK,Murphy KM,Sher A.Functional diversity of helper T lymphocytes.Nature,1996,383(6603):787-793.
21 Barten DM,Ruddle NH.Vascular cell adhesion molecule-1modulation by tumor necrosis factor in experimental allergic encephalomyelitis.J Neuroimmunol,1994,51(2):123-133
22 Issazadeh S,Mustafa M,Ljungdahl A,et al.Interferon gamma,interleukin 4 and transforming growth factor beta in experimental autoimmune encephalomyelitis in Lewis rats:dynamics of cellular mRNA expression in the central nervous system and lymphoid cells.J Neurosci Res,1995,40(5):579-590
23 Issazadeh S, Navikas V, Schaub M, et al. Kinetics of expression of costimulatory molecules and their ligands in murine relapsing experimental autoimmune encephalomyelitis in vivo. J Immunol, 1998, 161(3): 1104-1112
24 Begolka WS, Vanderlugt CL, Rahbe SM, et al.Differential expression of inflammatory cytokines parallels progression of central nervous system pathology in two clinically distinct models of multiple sclerosis. J Immunol, 1998, 161(8): 4437-4446
25 Chitnis T, Khoury SJ.Cytokine shifts and tolerance in experimental autoimmune encephalomyelitis. Immunol Res, 2003,28(3):223-239
26 Inobe J, Slavin AJ, Komagata Y, et al. IL-4 is a differenttiation factor for transforming growth factor-beta secreting Th3 cells and oral administration of IL-4 enhances oral tolerance in experimental allergic encephalomyelitis. Eur J Immunol, 1998,28(9): 2780-2790
27 Kuroda Y, Shimamoto Y. Human tumor necrosis factor-alpha augments experimental allergic encephalomyelitis in rats.J Neuroimmunol, 1991, 34(2-3): 159-164
28 Lauritsen JP, Christensen MD, Dietrich J, et al. Two distinct pathways exist for down-regulation of the TCR. J Immunol. 1998,161(1):260-267
29 Sela M. Immunomodulatory vaccines against autoimmune diseases.Rejuvenation Res, 2006, 9(1): 126-133
30 Butti E, Bergami A, Recchia A, et al. IL4 gene delivery to the CNS recruits regulatory T cells and induces clinical recovery in mouse models of multiple sclerosis.Gene Ther. 2008,15(7):504-515
1 Noseworthy JH,Lucchinetti C,Rodriguez M,et al Multiple Sclerosis[J].N Engl J Med,2000,343(13):938-952
2 莫晔,刘胜利.多发性硬化症20例临床分析[J].临床荟萃,1997,13(6):274-275
3 Hartung HP,Gonsette R,Konig N,et al.Mitoxantrone in progressive multiple sclerosis:a placebo-controlled,doubleblind,randomized,multicentre trial[J].Lancet,2002,360(9350):2018-2025
4 Patti F,Amato MP,Filippi M,et al.A double blind,placebocontrolled,phase Ⅱ,add-on study of cyclophosphamide(CTX)for 24 months in patients affected by multiple sclerosis on a background therapy with interferon-beta study denomination:CYCLIN[J].J Neurol Sci,2004,223(1):69-71
5 Achiron A,Kishner I,Sarova-Pinhas I,et al.Intravenous immunoglobulin treatment following the first demyelinating event suggestive of multiple sclerosis:a randomized,double-blind,placebo-controlled trial[J].Arch Neurol,2004,61(10):1515-1520
6 Blanco Y,Moral EA,Costa M,et al.Effect of glatiramer acetate(Copaxone) on the immuno- phenotypic and cytokine profile and BDNF production in multiple sclerosis:a longitudinal study [J].Neurosci Lett,2006,406(3):270-275
7 Martinelli-Boneschi F, Rovaris M, Johnson KP,et al.Effects of glatiramer acetate on relapse rate and accumulated disability in multiple sclerosisrmeta-analysis of three double-blind,randomized,placebo-controlled clinical trials [J]. Multiple Sclerosis,2003,9(4):349-355
8 Rubio-Terres C,Aristegui Ruiz I, Medina Redondo F,et al.Cost-utility analisys of multiple sclerosis treatment with glatiramer acetate or interferon beta in Spain[J]. Farm Hosp, 2003,27(3):159-165
9 Clanet M,Radue EW,Kappos L,et al.A randomized, double- blind,dose-comparison study of weekly interferon beta-1a in relapsing MS[J].Neurology,2002,59(10): 1507-1517
10 Coppola G,Lanzillo R,Florio C,et al.Long-term clinical experience with weekly interferon beta-1a in relapsing multiple sclerosis[J].Eur J Neurol,2006,13(9): 1014-1021
11 PRISMS(Prevention of Relapses and Disability by interferon beta-1a Subcutaneously in Multiple Sclerosis) Study Group and the University of British Columbia MS/MRI Analysis Group. PRISMS-4: Long-term efficacy of interferon-beta-1a in relapsing MS[J] .Neurology, 2001,56(12):1628-1636
12 European Study Group on interferon beta-1b in secondary progressive MS. Placebo-controlled multicentre randomised trial of interferon beta-1b in treatment of secondary progressive multiple sclerosis[J].Lancet, 1998,352(9139): 1491-1497
13 Vollmer T, Key L, Durkalski V, et al. Oral simvastatin treatment in relapsing-remitting multiple sclerosis[J].Lancet, 2004,363(9421): 1607-1608
14 Stuve O,Youssef S,Steinman L,et al. Statins as potential therapeutic agents in neuro-inflammatory disorders[J].Curr Opin Neurol,2003,16(3):393-401
15 Bielekova B,Catalfamo M,Reichert-Scrivner S,et al. Regulatory CD56(bright) natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis[J].Proc Natl Acad Sci USA,2006,103(15):5941-5946
16 Pluchino S,Quattrini A,Brambilla E,et al.Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis[J].Nature,2003,422(6933):688-694
17 Achiron A,Mandel M. T-cell vaccination in multiple sclerosis[J].Autoimmun Rev,2004,3(1): 25-32
18 Direskeneli H,Eksioglu-Demiralp E,Kibaroglu A,et al. Oligoclonal T cell expansions in patients with Behcet's disease[J].Clin Exp Immunol, 1999,117(1):166-170
19 Popovic N,Schubart A,Goetz BD,et al.Inhibition of autoimmune encephalomyelitis by a tetracycline[J].Ann Neurol, 2002,51(2):215-223
20 Metz LM,Zhang Y,Yeung M,et al.Minocycline reduces gadolinium-enhancing magnetic resonance imaging lesions in multiple sclerosis[J].Ann Neurol,2004,55(5):756