AChR α亚基基因片段联合IL-6 DNA免疫大鼠建立实验性重症肌无力模型

目的:探索用DNA免疫方法:建立实验性自身免疫性重症肌无力(EAMG)动物模型的可行性。方法:将含人乙酰胆碱受体α亚基N端主要免疫区205个氨基酸(AChRα205)的编码基因、含大鼠IL-6cDNA序列的基因分别亚克隆入真核表达载体pcDNA3.1(+)构建pcDNA3.1(+)/AChRα205、pcDNA3.1(+)/IL-6两种重组质粒。以pcDNA3.1(+)免疫对照组Lewis大鼠,pcDNA3.1(+)/AChRα205和pcDNA3.1(+)/AChRα205加pcDNA3.1(+)/IL-6分别免疫两个实验组大鼠。从临床症状、肌电图、血清抗AChR抗体、组织病理与超微结构、组织基因整合和RNA转录几方面进行检测与评估。结果:实验组动物出现了临床肌无力症状;重复神经电刺激呈衰减反应;血清抗AChR抗体滴度增高;组织学与超微结构检查呈退行性改变;这些变化在双质粒共注射组更明显。两实验组大鼠均有目的:基因的整合与转录。结论:用重组质粒pcDNA3.1(+)/AChRα205、pcDNA3.1(+)/IL-6免疫Lewis大鼠建立了EAMG模型,方法:简便实用。     

重症肌无力实验模型中胸腺细胞凋亡及其机制的研究

为观察重症肌无力中胸腺细胞凋亡的变化 ,制备了实验性自身免疫性重症肌无力 (EAMG )模型 ,采用切口及末端标记 (TUNEL )的方法观察了病程的不同阶段中胸腺细胞的凋亡情况。结果表明 ,EAMG的胸腺出现了细胞凋亡指数的减少 ,在免疫后第 4周 ,细胞凋亡指数减少最明显 (P <0 0 1)。进一步观察 ,可见实验鼠从第 4周开始 ,EAMG胸腺中Bcl 2的表达增加 ,而且细胞凋亡指数的减少与Bcl 2表达分数的增加呈明显的负相关。由此可见Bcl 2表达的增加 ,可导致EAMG胸腺中激活免疫细胞的正常凋亡过程抑制 ,可能是MG自身免疫发病机制之一。     

升陷汤治疗实验性自身免疫性重症肌无力大鼠免疫机制研究

目的:探讨升陷汤治疗实验性自身免疫性重症肌无力(EAMG)大鼠的免疫机制。方法:采用人工合成鼠源乙酰胆碱受体琢亚基97鄄116 肽段加完全弗氏佐剂免疫Lewis 大鼠构建EAMG,经评估确定建模成功25 只。将这25 只大鼠随机分为模型对照组,阳性药物组(强的松组),升陷汤低、中、高剂量组。观察该方对模型大鼠临床评分、体重、低频重复电刺激(RNS,5 Hz)衰减率、血清中乙酰胆碱抗体(AChR Ab)含量及TGF、IFNβ、IL-2、IL-4 和IL-17 水平的影响。结果:造模后各组较佐剂对照组RNS 衰减率明显增大(P<0.01 或P<0.05),且均超过10%,体重进行性下降,并出现典型肌无力样症状,证明造模成功。给药治疗后,升陷汤低、中、高剂量组大鼠RNS 衰减率均显著降低(P<0.01),体重下降均减缓,症状好转。与模型对照组相比,升陷汤低、中、高剂量组血清AChR Ab 含量均降低(P<0.05),TGFβ水平均升高,IFN、IL-2、IL-4 和IL-17 水平均降低(P<0.01 或P<0.05)。结论:升陷汤可使RNS 衰减好转,改善EAMG 大鼠体重持续下降趋势,并使体重增加趋势升高,其机制可能是上调TGFβ1水平,下调IFN、IL-2、IL-4 和IL-17 的水平,抑制B 细胞产生AChR Ab,降低血清AChR Ab 含量,减少对NMJ 处AChR 的损害,从而起到治疗EAMG 的作用。     

NK1.1+细胞在实验性重症肌无力发病机制中的作用

目的：探讨NK1．1^＋细胞在重症肌无力疾病进展中发挥的作用以及其调节机制。方法：（1）用腹腔注射抗NK1．1抗体和抗TGF—β抗体的方法制造NK1．1^＋细胞缺陷和TGF—β缺陷的小鼠模型；（2）通过肌无力评分标准来评价试验组和对照组小鼠之间实验性自身免疫性重症肌无力（EAMG）的发病情况和病情严重程度的区别；（3）观察淋巴细胞增殖以及应用ELISA方法检测细胞因子TGF—β、抗AchRIgG的各种亚型。结果：（1）NK1．1^＋细胞缺陷的小鼠同对照组小鼠相比EAMG发病时间延迟且病情严重程度明显降低，但在免疫后7天用抗NK1．1处理却不能改变EAMG的临床病程。（2）NK1．1^＋细胞缺陷没有明显改变由AchR刺激产生的T细胞增殖反应，但由AchR特异性T细胞所产生的TGF—β有明显的增高。（3）经TGF—β中和抗体处理的NK1．1^＋细胞缺陷的小鼠其EAMG的进展明显加快。（4）NK1．1^＋细胞缺陷鼠血清中抗AchRIgG、AchRIgG2b亚型的含量下降。结论：NK1．1^＋细胞在EAMG的起始时发挥重要的作用，并且NK1．1^＋细胞的缺失将导致AchR特异性T细胞产生TGF-β的增加，从而抑制了重症肌无力的进展。     

AChR单抗(A7)诱导大鼠了实验性重症肌无力的分子基础

乙酰胆碱受体（ＡＣｈＲ）抗体在重症肌无力（ＭＧ）和实验性自身免疫性重症肌无力（ＥＡＭＧ）患者体内可诱导ＡＣｈＲ丢失和肌力减退。通过对小鼠抗电鳐电器官的ＡＣｈＲ单抗Ａ７的致病性测定和可变区基因克隆与序列分析，　探讨致病性抗体介导的ＭＧ和ＥＡＭＧ的发病机制。Ａ７被动注射大鼠后可诱导出严重的ＥＡＭＧ，ＡＣｈＲ损失串达３８．４％±７．２％。Ａ７的Ｈ链Ｖ区由小鼠ＰＣ７１８３胚系基因编码与Ｄ和ＪＨ４连接，与ＤＦＬ１６．２胚系Ｖ_Ｈ基因具有９３．７％的同源性。Ｌ链Ｖ区由小鼠Ｖ_Ｋ３组基因编码，与Ｊｋ２连接，与Ｖｋ２１Ｅ胚系基因具有９８．１％的同源性。     

受体相关蛋白对实验性自身免疫性重症肌无力的乙酰胆碱受体的作用

目的：探讨受体相关蛋白(Rapsyn)对实验性自身免疫性重症肌无力(EAMG)动物终板乙酰胆碱受体的作用。方法：用基因工程的方法制备提取pcDNA-Rapsyn，并将其注入动物肌肉左大腿外侧，右大腿相应部位注射相同剂量的空白质粒(Vector)，以方波刺激使其进入细胞膜内，2周后用单克隆抗体35(mAb35)诱导出EAMG模型并进行症状评估，mAb35诱导48小时后取双大腿外侧肌，用放射免疫法检测AChR，并计算出AChR的损失率。结果：在EAMG模型中，被pcDNA-Rapsyn预处理的肌肉AChR损失率明显低于未经处理的肌肉。结论：Rapsyn蛋白对EAMG模型的AChR有保护作用。     

细胞因子在重症肌无力发病机制中的研究进展

重症肌无力(MG)主要是由乙酰胆碱受体抗体(AChR-Ab)介导,依赖细胞免疫和补体参与的使神经肌肉接头处突触后膜上乙酰胆碱受体受损的自身免疫性疾病.近年来的研究表明,细胞因子间的平衡紊乱与MG的发病有重要关系.因而深入了解不同细胞因子在MG中的作用可以更加明确MG的发病机制及对其治疗提出新的方案.     

重肌灵片治疗重症肌无力的免疫调节机理研究

目的：探讨重肌灵片的免疫调节机制。方法：采用正常小鼠、免疫抑制模型小鼠和EAMG大鼠模型观察重肌灵片的免疫调节作用。结果：重肌灵片增强ConA或LPS诱导的正常小鼠及免疫抑制模型小鼠T、B细胞的增殖，促进1L-2分泌；能对抗ConA诱导的EAMG大鼠淋巴细胞增殖的降低；但抑制AChR诱导的淋巴细胞增殖及IFN-γ、IL-4mRNA的表达；此外，能显著促进EAMG大鼠CD4^＋T淋巴细胞的凋亡。结论：重肌灵片增强正常小鼠、免疫抑制小鼠和EAMG大鼠的免疫功能，但抑制AChR诱导的特异性的T细胞增殖及IFN-γ、IL4mRNA的表达，该作用可能是通过诱导AChR特异性的CD4^＋T细胞凋亡实现的。     

乙酰胆碱受体制剂对实验性自身免疫性重症肌无力的抑制作用

在采用乙酰胆碱受体免疫Ｌｅｗｉｓ大鼠诱发实验性自身免疫性重症肌无力（ＥＡＭＧ）前，让动物口服ＡｃｈＲ产生免疫耐受，并采用ＥＬＩＳＡ测定血清抗ＡｃｈＲＩｇＧ抗体水平和放射免疫法测定肌肉ＡｃｈＲ含量。结果显示能预防ＥＡＭＧ临床发病，明显地抑制血清抗ＡｃｈＲＩｇＧ抗体反应，并减少肌肉ＡｃｈＲ损失。本研究结果表明口服ＡｃｈＲ产生免疫耐受是一种简单、有效、特异性抑制ＥＡＭＧ的方法。     

眼肌型重症肌无力免疫学机制研究进展

眼肌型重症肌无力(ocular myasthenia gravis,oMG)是指肌无力症状仅局限于眼外肌,以单侧或双侧上眼睑下垂、复视、易疲劳等为主要临床表现的自身免疫性疾病,其免疫学机制复杂,由多种致病性抗体、细胞因子参与及不同补体激活途径共同发挥作用。文章就近年来oMG免疫学发病机制的研究进展作一综述。     

Neuromuscular transmission in experimental autoimmune myasthenia gravis (EAMG)

Chronic experimental autoimmune myasthenia gravis (EAMG) was induced in rats by immunization with acetylcholine receptor (AChR) purified from the electroplax of Torpedo californica. 35–40 days after immunization, serum anti-AChR antibody titers were about 40 nM. At this stage, electrophysiology was performed on isolated M. omohyoideus muscle-preparations from myasthenic and from normal (control) rats.For the study of the equilibrium interaction between acetylcholine (ACh) and AChR, dose-response curves were obtained by quantitative ionophoretic application of ACh to voltage-clamped end-plates. Analysis of dose-response curves yielded the following parameters: maximum end-plate conductance per unit surfaceg _max (EAMG)=10.3±1.1 nS/m²,g _max (normal)=20.2±1.8 nS/m²; apparent dissociation constant K (EAMG)=96±5 M, K (normal)=58±6 M; Hill-coefficient n_H (EAMG)=2.3±0.1, n_H (normal)=2.3±0.1. Single channel properties were derived from an analysis of ACh-induced end-plate current noise: the mean single channel conductance was (EAMG)=29.1±2.2 pS, (normal)=27.6±1.8 pS and the mean channel life-time (EAMG)=1.39±0.09 ms, (normal)=1.32±0.08 ms (T=22°C).The electrophysiological data are interpreted as follows: (1) At myasthenic end-plates there is a 50–60% reduction of functioning AChR (decrease ofg _max). A total number of about 2×10⁶ (1×10⁶) channels per end-plate was calculated for control (myasthenic) rats. (2) The affinity of AChR for ACh is reduced and/or there is an impediment of the conformational change from the closed- to the open-channel configuration (increase of K). (3) Single channel properties are essentially unaffected.This work was supported by the Deutsche Forschungsgemeinschaft SFB 38, project N and We 667/6     

Differential requirements for CD28 and CD40 ligand in the induction of experimental autoimmune myasthenia gravis

The interactions of CD28-B7 and CD40-CD40 ligand (CD40L) pathways in T cell co-stimulation and autoimmune disease are incompletely understood. We sought to address this issue by investigation of the genesis of acetylcholine receptor (AChR)-induced antibody-mediated experimental autoimmune myasthenia gravis (EAMG) in CD28- and CD40L-deficient mice (CD28^{− / −} , CD40L^{− / −} ). Compared to wild-type mice, the CD28^{− / −} mice became less susceptible, and CD40L^{− / −} mice were completely resistant to EAMG induction. Analysis of T helper functions, reflected by cytokine responses, revealed a switch to a Th1 profile in CD28^{− / −} mice. Consistently, levels of serum AChR-specific antibodies of the IgG1 isotype were decreased in CD28^{− / −} mice. In the CD40L^{− / −} mice, both Th1 and Th2 cytokine responses were diminished, and T cell-dependent AChR-reactive B cell responses were more severely impaired than in the CD28^{− / −} mice. Thus, CD28 and CD40L are differentially required for induction of EAMG.     

Nasal tolerance in experimental autoimmune myasthenia gravis (EAMG): induction of protective tolerance in primed animals

Nasal administration of μg doses of acetylcholine receptor (AChR) is effective in preventing the development of B cell-mediated EAMG in the Lewis rat, a model for human MG. In order to investigate whether nasal administration of AChR modulates ongoing EAMG, Lewis rats were treated nasally with AChR 2 weeks after immunization with AChR and Freund's complete adjuvant. Ten-fold higher amounts of AChR given nasally (600 μg/rat) were required to ameliorate the manifestations of EAMG compared with the amounts necessary for prevention of EAMG. In lymph node cells from rats receiving 600 μg/rat of AChR, AChR-induced proliferation and interferon-gamma (IFN-γ) secretion were reduced compared with control EAMG rats receiving PBS only. The anti-AChR antibodies in rats treated nasally with 600 μg/rat of AChR had lower affinity, reduced proportion of IgG2b and reduced capacity to induce AChR degradation. Numbers of AChR-reactive IFN-γ and tumour necrosis factor-alpha (TNF-α) mRNA-expressing lymph node cells from rats treated nasally with 600 μg/rat of AChR were suppressed, while IL-4, IL-10 and transforming growth factor-beta (TGF-β) mRNA-expressing cells were not affected. Collectively, these data indicate that nasal administration of AChR in ongoing EAMG induced selective suppression of Th1 functions, i.e. IFN-γ and IgG2b production, but no influence on Th2 cell functions. The impaired Th1 functions may result in the production of less myasthenic anti-AChR antibodies and contribute to the amelioration of EAMG severity in rats treated with AChR 600 μg/rat by the nasal route.     

SIN-1降低重症肌无力大鼠的自身免疫反应

用一氧化氮 (NO)供体 3 吗啉 斯德酮亚胺 (SIN 1)干预乙酰胆碱受体 (AChR)致敏的实验性自身免疫性重症肌无力 (EAMG)大鼠模型。并进行临床评分、体重测量、免疫学指标及细胞凋亡检测。结果显示在大鼠致敏后 0～ 7天和 16～ 2 5天给药后 ,SIN 1组大鼠临床症状明显减轻伴发病延迟 ,血清IgG含量始终明显低于对照组 ;致敏后第10天 ,SIN 1组大鼠脾单个核细胞AChR反应性IFN γ分泌性细胞数 (6 38± 1 0 6 )比对照组 (8 2 5± 1 6 7)明显减少(P <0 0 5 ) ,外周血AChR反应性凋亡细胞含量 (8 5 9± 1 35 )与对照组 (5 0 5± 0 6 3)相比明显增多 (P <0 0 1)。实验证明SIN 1可以促使AChR反应性MNC凋亡增加 ,下调自身免疫反应 ,在EAMG的发病和疾病进展中起保护作用。     

Dehydroepiandrosterone therapy ameliorates experimental autoimmune myasthenia gravis in Lewis rats

To detect a possible effect of dehydroepiandrosterone (DHEA) in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), DHEA (0.5 mg/rat) was administrated intraperitoneally to Lewis rats every other day from day 4 postimmunization (p.i.) to day 35 p.i. with Torpedo acetylcholine receptor (AChR) and Freund's complete adjuvant. Rats treated with DHEA had a lower clinical score (mean clinic score, 2 versus 0.5 on day 37 p.i.) and a lower body weight loss (mean body weight, 169 versus 142 g on day 37 p.i.) compared with control EAMG rats. DHEA treatment decreased serum anti-AChR IgG and IgG2b antibody titers on days 7, 14, and 21 p.i. and inhibited the levels of anti-AChR IgG antibody secreting cells (60%), accompanied by decreased IL-4 (33%) and augmented TGF-1-positive cells (41%) among lymph node mononuclear cells. These results obtained from EAMG in Lewis rats further encourage us to study DHEA treatment in human MG.     

Activation of the receptor for advanced glycation end products (RAGE) exacerbates experimental autoimmune myasthenia gravis symptoms

RAGE belongs to immunoglobulin superfamily and serves as a ligand for various immunoregulatory molecules including S100B that has been demonstrated important to T cell mediated autoimmune diseases. In this context, we hypothesized that RAGE could also impact B cell mediated, T cell-dependent autoimmune diseases. This was tested using myasthenia gravis (MG) animal model, EAMG. We show that expression of both RAGE and S100B are increased during EAMG and the interaction between RAGE and S100B affected the Th1/Th2/Th17/Treg cell equilibrium, up-regulate AChR-specific T cell proliferation. Furthermore, addition of S100B in vitro stimulated splenocyte activity linked to COX-2 up-regulation. NS-398, a selective COX-2 inhibitor, effectively diminished S100B mediated activity of AChR-specific antibody secreting splenocytes. These findings suggested that a reciprocal relationship between RAGE and S100B promoted the development of EAMG, highlighting the importance of understanding the mechanisms of EAMG disease as a means of developing new therapies for the treatment of MG.     

Age- and sex-related resistance to chronic experimental autoimmune myasthenia gravis (EAMG) in Brown Norway rats

The influence of age and sex on the induction of chronic EAMG was analysed. Aged male rats, immunized with Torpedo acetylcholine receptor (tAChR), showed no clinical signs of disease or AChR loss. Immunization of young male Brown Norway (BN) rats resulted in both clinical signs of disease and 65% AChR loss. In contrast, both young and aged female BN rats showed comparable AChR loss (58% and 50%, respectively), although aged female rats did not develop clinical signs of disease. Differences in antibody titres, isotype distribution, fine specificity or complement activation could not account for the observed resistance. These results suggest that resistance against EAMG in aged rats is due to resistance of the AChR against antibody-mediated degradation, or to mechanisms able to compensate for AChR loss.     

Complement and cytokine based therapeutic strategies in myasthenia gravis

Myasthenia gravis (MG) is a T cell–dependent and antibody-mediated disease in which the target antigen is the skeletal muscle acetylcholine receptor (AChR). In the last few decades, several immunological factors involved in MG pathogenesis have been discovered mostly by studies utilizing the experimental autoimmune myasthenia gravis (EAMG) model. Nevertheless, MG patients are still treated with non-specific global immunosuppression that is associated with severe chronic side effects. Due to the high heterogeneity of AChR epitopes and antibody responses involved in MG pathogenesis, the specific treatment of MG symptoms have to be achieved by inhibiting the complement factors and cytokines involved in anti-AChR immunity. EAMG studies have clearly shown that inhibition of the classical and common complement pathways effectively and specifically diminish the neuromuscular junction destruction induced by anti-AChR antibodies. The inborn or acquired deficiencies of IL-6, TNF-α and TNF receptor functions are associated with the lowest EAMG incidences. Th17-type immunity has recently emerged as an important contributor of EAMG pathogenesis. Overall, these results suggest that inhibition of the complement cascade and the cytokine networks alone or in combination might aid in development of future treatment models that would reduce MG symptoms with highest efficacy and lowest side effect profile.     

Treatment of passively transferred experimental autoimmune myasthenia gravis using papain

Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p. injection of anti-MIR mAb35. A total of 0.75 mg of papain was given as one or three injections 3-7 h after MoAb injection. The mAb35 + papain-treated animals developed mild weakness during the first 30 h and subsequently recovered, while all animals that received only mAb35 developed severe myasthenic symptoms and died within 24-30 h. Animals treated only with papain showed no apparent side effects for up to 2 months. Serum anti-AChR levels in mAb35 + papain-treated rats decreased within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle AChR in mAb35 + papain-treated animals was partially protected from antibody-mediated degradation. These results show that treatment of rats with papain can prevent passively transferred EAMG without any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in myasthenia gravis.