黄芪复方治疗重症肌无力的临床与实验研究
|
摘要
重症肌无力(myasthenia gravis,MG)是一种主要累及神经肌肉接头处(neuromuscular junction,NMJ)突触后膜(postsynaptic)上乙酰胆碱受体(acetylchol ine receptor,AchR),主要由乙酰胆碱受体抗体(acetylcholinereceptorantibody,AchRab)介导、细胞免疫(cell-mediated immunity,CMI)依赖性、补体(complement)参与的自身免疫性疾病(autoimmune disease)。其特点包括:1.临床上表现为活动后加重、休息后减轻、朝轻暮重的骨骼肌无力。2.电生理上表现为低频重复电刺激波幅递减,微小终板电位降低、单纤维肌电图上颤抖增宽、甚至阻滞。3.药理学上表现为胆碱酯酶抑制剂治疗有效、对箭毒类药物的过度敏感性。4.免疫学上表现为血清乙酰胆碱受体抗体增高。5.免疫病理学上表现为神经肌肉接头处突触后膜的皱褶减少,变平坦,突触后膜上乙酰胆碱受体减少。重症肌无力是一种世界性疑难病,目前西医治疗方法均有不同程度的副作用,因此,发掘减少西药副作用且疗效确切的中药成为当务之急。黄芪复方颗粒剂是导师张静生教授在脾肾学说指导下研制成功的治疗重症肌无力的纯中药制剂,在临床和实验研究中均取得了明显疗效。
目的:观察黄芪复方治疗重症肌无力的临床疗效;观察黄芪复方对重症肌无力患者外周血淋巴细胞亚群:辅助性淋巴细胞亚群Th1/Th2、抑制性淋巴细胞亚群Tc1/Tc2;细胞因子IFN-γ、IL-4及TNF-α水平;免疫球蛋白IgG、IgA、IgM、补体C3、C4和AChRAb水平的影响,从细胞水平揭示中药黄芪复方治疗MG的免疫机理。
方法:
1.根据诊治先后,将符合纳入标准的重症肌无力患者随机分为治疗组和对照组,两组各30例。治疗组服用黄芪复方颗粒剂和强的松;对照组服用强的松和模拟剂。90天为一疗程。观察治疗前后的临床疗效积分和中医证候积分变化。
2.应用流式细胞仪检测治疗前后的重症肌无力患者外周血淋巴细胞亚群分布的变化情况。
3.应用流式细胞仪检测黄芪复方颗粒剂及模拟剂和强的松治疗前后重症肌无力患者外周血Th1、Tc1、Th2、Tc2细胞的变化情况。
4.采用酶联免疫吸附实验技术检测重症肌无力患者治疗前后外周血血清细胞因子IFN-γ、IL-4及TNF-α水平的变化。
5.采用免疫比浊及酶联免疫吸附实验技术检测重症肌无力患者治疗前后外周血IgG、IgA、IgM、C3、C4、AChRAb水平的变化。
结果:
1.临床研究结果显示:黄芪复方对重症肌无力具有明显疗效。从临床疗效积分变化统计:治疗组显效率70.0%,对照组显效率56.7%,二者相比较,治疗组疗效优于对照组,具有显著性差异(P＜0.05);从中医证候积分变化统计:治疗组有效率73.33%,对照组有效率仅为56.67%,两组中医证候改善情况有显著差异,P＜0.01,提示治疗组在中医证候改善方面明显优于对照组。研究还发现,对照组治疗后出现轻度肝功能异常、空腹血糖增高、向心性肥胖等副作用;而中药黄芪复方具有确切的治疗作用,且未发现任何毒副作用,明显优于对照组。
2.两组患者治疗前外周血CD4~+细胞及CD4~+/CD8~+比值均明显高于健康组,差异均有统计学意义(P＜0.01)。CD8~+细胞、NK细胞(CD16~+/56~+)水平较健康组显著降低,差异有统计学意义(P＜0.01),两组CD3~+、总B细胞(CD19~+)比较无统计学差异(P＞0.05)。治疗后,两组CD3~+细胞无明显变化(P＞0.05),而CD4~+细胞、CD4~+/CD8~+比值及总B细胞(CD19~+)与治疗前比较均有明显下降,CD8~+及NK细胞(CD16~+/56~+)均明显增加,差异均有统计学意义(P＜0.05)。与对照组治疗后相比较治疗组CD4~+、CD8~+、CD4~+/CD8~+、CD19~+、CD16~+/56~+均有显著差异(P＜0.05)。
3.与健康组相比较,两MG组外周血中Th1、Th2、Tc1和Tc2细胞百分数显著增高,差异有统计学意义(P＜0.01),Th1/Th2、Tc1/Tc2比值同健康组相比差异有统计学意义(P＜0.05)。治疗后,治疗组Th1型、Tc1型细胞明显减少(P＜0.01),与对照组治疗后比较有显著性差异(P＜0.05)。
4.两MG组外周血IFN-γ、IL-4、TNF-α水平均明显高于健康组,差异均有统计学意义(P＜0.01)。与治疗前相比较,两MG组经治疗后IFN-γ、IL-4、TNF-α水平均显著降低,差异均有统计学意义(P＜0.01)。两组治疗后比较,治疗组各项指标下降显著,与对照组比较,差异均有统计学意义(P＜0.05)。
5.与健康组比较,两MG组AchRab水平显著增高,差异有统计学意义(P＜0.01)。两MG组IgA、IgM水平均显著低于健康组,差异有统计学意义(P＜0.01)。两MG组C_3、C_4水平显著降低,与健康组比较差异有统计学意义(P＜0.05)。经治疗后,治疗组补体C_3、C_4水平与治疗前相比显著增高,差异均有统计学意义(P＜0.05)。治疗组AchRab水平与治疗前比较有明显下降,差异有统计学意义(P＜0.01)。治疗组补体C_3、C_4及AchRab水平与对照组治疗后比较,差异有统计学意义(P＜0.05)。
结论:
1.黄芪复方+基础西药能明显改善重症肌无力患者的临床症状和中医证候,在中医证候改善方面明显优于单独应用强的松治疗,而且无任何毒副作用。
2.黄芪复方可能通过抑制Th细胞增殖,促进T细胞活化,降低CD4~+/CD8~+,使重症肌无力患者体内T淋巴细胞亚群比例重新分布,纠正重症肌无力异常的免疫应答过程,最终抑制B细胞产生以乙酰胆碱受体抗体为主的自身抗体。
3.黄芪复方通过抑制MG患者Th1型细胞和Tc1型细胞的分化,减少Th1/Tc1型细胞因子如IFN-γ的分泌而起到抗炎和改变病程的作用。
4.经过黄芪复方治疗后,血清中IFN-γ、IL-4、TNF-α均有不同程度的降低,可以反映中药治疗MG的免疫效应。在免疫抑制方面,黄芪复方+免疫抑制剂强的松治疗组比单用免疫抑制剂强的松效果明显,说明黄芪复方对调节免疫功能是双向的、多靶点的,而强的松对病人的免疫功能是过度抑制的,这显示出中药制剂黄芪复方的整体调节特征和优势。
5.黄芪复方规范化治疗以后MG患者血清中的补体C3、C4明显增高提示对MG患者测定血清补体,有助于进行动态观察,并可作为判断病情严重程度(活动性)的客观指标。
Myasthenia gravis is a kind of autoimmune disease,it mainly involves the Acetylcholine receptor(AchR) on the postsynaptic membrane of neuromuscular junction,mediated by the specific nicotinic acetylcholine receptor(N_2AchRab), and the complement participates its process.Its characteristics involve:1.Clinical symptoms:weakness of skeletat muscle,aggravated after action,and relieved after rest,slight in daytime and gravis at night.2.Electromyography:the amplitude decreased after echo-low frequency transduction of electric stimulation,at the same time,the miniature end-plate potential decreased,and the jitter of single fiber EMG widened,even blocked.3.Pharmacology:treated with cholinesterase inhibitor is effective, and over anaphylaxis to tubocurarine.4.Immunology:the increasing of acetylcholine receptor antibody in blood serum.5,Immunopathology:the wrinkles of postsynaptic membrane at neuromuscular junction decreased,flattened,and the AchR on the postsynaptic membrane decreased,myasthenia gravis is a cosmopolitan stubborn disease,the western medical treatment has its side effect, therefore,develop the effective Chinese patent medicine is the urgent mission. the complex recipe of Astragalus membranaceus is a pure Chinese drugs pharmaceutics,developed by Pro.Zhang Jingsheng under the directions of PISHEN theory,and obtained manifest therapeutic effect on the clinical and experimental study till now.
Purpose:Observe the complex recipe of Astragalus membranaceus's clinical effect in treating Myasthenia graves,provide its experimental basement;Observe the influence of the complex recipe of Astragalus membranaceus to T lymphocyte、Tc1/Tc2、Th1/Th2、IFN-γ、IL-4、TNF-α、IgG、IgA、IgM、C3、C4、AchRAb in peripheral blood of myasthenia gravis patients,reveal its immunological regulation effect on the cellulax level.
Methods:
1.According to the hospitalization date and the random-double blind-control principle,the myasthenia gravis patients was randomly divided into 2 groups, the curing group and the control group,30 cases per group.Patients of curing group administered the complex recipe of Astragalus membranaceus and prednisone placebo,Patients of control group administered prednisone tablet and Zhongjiling tablet placebo.Course of treatment is 90 days.Compare the pre-treatment and post-treatment clinical curative effect scores and Chinese medicals symptom scores.
2.Detect the pre-treatment and post-treatment distribution of T lymphocyte subgroup of myasthenia gravis patients with flow cytometry.
3.Detect the pre-treatment and post-treatment distribution of T lymphocyte subgroup Th1、Tc1、Th2、Tc2 of myasthenia gravis patients with flow cytometry.
4.Detect the the pre-treatment and post-treatment level of IFN-γ、IL-4、TNF-αof myasthenia gravis patients with ELISA assay technology。
5.Using the immunoturbidimetric and ELISA assay technology to detect the level of IgG、IgA、IgM、C3、C4、AChRAb in patients' peripheral blood pre-and post-administration.
Results:
1.Clinical research:The complex recipe of Astragalus membranaceus has significant effect to myasthenia gravis,the clinical syndrome score statistic result shows that the effective ratio of curing group is 70.0%,and that if control group is 56.7%,manifest difference exit between these two groups, P＜0.05;the change of Chinese medical symptom score statistic result shows that the effective ratio of curing group is 73.33%,and that if control group is 56.67%,manifest difference exit between these two groups,P＜0.01,it indicate that the curing group is better than that control group in improving Chinese medical symptoms.In this study,the patients in control group appeared side-effects such as sight hepatic dysfunction,increasing fasting serum glucose,femoral head necrosis and central obesity,on the contrary,The complex recipe of Astragalus membranaceus has exact curing effect without any side-effect,these shows that it is better than control group.
2.Before treatment,the CD4~+ cell percentage and CD4~+/CD8~+ of these two groups are higher than that of normal group,P＜0.01,the CD8~+ and NK(CD16~+/56~+)cell percentage of these two groups are lower than that of normal group,P＜0.01.The CD3~+ and B(CD19~+) cell percentage of these two groups have no difference with that of normal group,P＞0.05.After treatment,there is no change on the CD3~+ cell percentage of these two groups,P＞0.05,CD4~+ and CD19~+ cell percentage and the ratio of CD4~+/CD8~+ decreased significantly,CD8~+ and NK(CD16~+/56~+) cell increased significantly,P＜0.05.Compare with that of control group,there is significant difference for the CD4~+,CD8~+,CD4~+/CD8~+,CD19~+ and CD16~+/56~+ cell percentage,P＜0.05.
3.The Th1,Th2,Tc1 and Tc2 cell percentage of these two groups are higher than that of normal group,P＜0.01,Th1/Th2 and Tc1/Tc2 have difference with that of normal group,P＜0.05.After treatment,The Th1 and Tc1 cell percentage of the curing group decreased significantly,P＜0.01,there is obvious difference with that of control group.
4.The IFN-γ,IL-4 and TNF-αlevel of these two groups are significant higher than that of normal group,P＜0.01.After treatment,the IFN-γ,IL-4 and TNF-αlevel of two groups depressed,compared with that of pre-treatment, significant difference exited,P＜0.01.While in control group,the IFN-γ,IL-4 and TNF-αlevel decreased significantly,there is obvious difference with that of control group,P＜0.05.
5.The AchRab level of these two groups are higher than that of normal group, P＜0.01.The IgA and IgM level of these two groups are lower than that of normal group,P＜0.01.The C_3 and C_4 level of these two groups decreased significantly, there is obvious difference with that of normal group,P＜0.05.After treatment, the C_3 and C_4 level of curing group increased,compared with that of pre-treatment,significant difference exited,P＜0.05,the AChRAb level of the curing group decreased significantly,P＜0.01,while in control group,the C_3, C_4 and AchRab level are obvious difference with that of control group,P＜0.05.
Conclusion:
1.The complex recipe of Astragalus membranaceus can significantly improve the myasthenia gravis pationts' clinical symptoms and Chinese medicine syndromes,Its effect of improving Chinese medicine symptoms is better than that of only using prednisone,and no adverse reaction exited in this study.
2.The complex recipe of Astragalus membranaceus caused the redistribution of T lymphocyte subgroup ratio,this effect maybe due to its inhibition of Th cell proliferation,promoting Ts cell's activation and depress CD4~+/CD8~+ ratio, then rectified the abnormal immune response of myasthenia gravis patients, as a result,inhibited the production of auto antibody,mainly AchRab.
3.Th1 and Tc1 subsets decreased in the peripheral blood after MG patients were treated with leflunomide.The effect of leflunomide might be related to the prevention of Th1 and Tc1 differentiation and the decrease of Th1 and Tc1 cytokines such as IFN-γ.
4.After treatment with the complex recipe of Astragalus membranaceus,the IFN-γ, IL-4 and TNF-αlevel depressed,compared with that of pre-treatment,this indicated its immunological regulation effect.The complex recipe of Astragalus membranaceus+ prednisone has exact curing effect,it is better than that of only using prednisone.These indicate that The complex recipe of Astragalus membranaceus's immunological regulation is multi-targets and tow-way regulation.It is different with that of western medicine,which is overall inhibition to patients' immune function,and this manifest The complex recipe of Astragalus membranaceus's holo-regulation characteristic and predominance.
5.After treatment,the C_3 and C_4 level of curing group increased,it suggests that defecting of the levels of the complements in MG patients has attributions to mobile MG and maybe an objective index in determing the disease severity.
目的:观察黄芪复方治疗重症肌无力的临床疗效;观察黄芪复方对重症肌无力患者外周血淋巴细胞亚群:辅助性淋巴细胞亚群Th1/Th2、抑制性淋巴细胞亚群Tc1/Tc2;细胞因子IFN-γ、IL-4及TNF-α水平;免疫球蛋白IgG、IgA、IgM、补体C3、C4和AChRAb水平的影响,从细胞水平揭示中药黄芪复方治疗MG的免疫机理。
方法:
1.根据诊治先后,将符合纳入标准的重症肌无力患者随机分为治疗组和对照组,两组各30例。治疗组服用黄芪复方颗粒剂和强的松;对照组服用强的松和模拟剂。90天为一疗程。观察治疗前后的临床疗效积分和中医证候积分变化。
2.应用流式细胞仪检测治疗前后的重症肌无力患者外周血淋巴细胞亚群分布的变化情况。
3.应用流式细胞仪检测黄芪复方颗粒剂及模拟剂和强的松治疗前后重症肌无力患者外周血Th1、Tc1、Th2、Tc2细胞的变化情况。
4.采用酶联免疫吸附实验技术检测重症肌无力患者治疗前后外周血血清细胞因子IFN-γ、IL-4及TNF-α水平的变化。
5.采用免疫比浊及酶联免疫吸附实验技术检测重症肌无力患者治疗前后外周血IgG、IgA、IgM、C3、C4、AChRAb水平的变化。
结果:
1.临床研究结果显示:黄芪复方对重症肌无力具有明显疗效。从临床疗效积分变化统计:治疗组显效率70.0%,对照组显效率56.7%,二者相比较,治疗组疗效优于对照组,具有显著性差异(P＜0.05);从中医证候积分变化统计:治疗组有效率73.33%,对照组有效率仅为56.67%,两组中医证候改善情况有显著差异,P＜0.01,提示治疗组在中医证候改善方面明显优于对照组。研究还发现,对照组治疗后出现轻度肝功能异常、空腹血糖增高、向心性肥胖等副作用;而中药黄芪复方具有确切的治疗作用,且未发现任何毒副作用,明显优于对照组。
2.两组患者治疗前外周血CD4~+细胞及CD4~+/CD8~+比值均明显高于健康组,差异均有统计学意义(P＜0.01)。CD8~+细胞、NK细胞(CD16~+/56~+)水平较健康组显著降低,差异有统计学意义(P＜0.01),两组CD3~+、总B细胞(CD19~+)比较无统计学差异(P＞0.05)。治疗后,两组CD3~+细胞无明显变化(P＞0.05),而CD4~+细胞、CD4~+/CD8~+比值及总B细胞(CD19~+)与治疗前比较均有明显下降,CD8~+及NK细胞(CD16~+/56~+)均明显增加,差异均有统计学意义(P＜0.05)。与对照组治疗后相比较治疗组CD4~+、CD8~+、CD4~+/CD8~+、CD19~+、CD16~+/56~+均有显著差异(P＜0.05)。
3.与健康组相比较,两MG组外周血中Th1、Th2、Tc1和Tc2细胞百分数显著增高,差异有统计学意义(P＜0.01),Th1/Th2、Tc1/Tc2比值同健康组相比差异有统计学意义(P＜0.05)。治疗后,治疗组Th1型、Tc1型细胞明显减少(P＜0.01),与对照组治疗后比较有显著性差异(P＜0.05)。
4.两MG组外周血IFN-γ、IL-4、TNF-α水平均明显高于健康组,差异均有统计学意义(P＜0.01)。与治疗前相比较,两MG组经治疗后IFN-γ、IL-4、TNF-α水平均显著降低,差异均有统计学意义(P＜0.01)。两组治疗后比较,治疗组各项指标下降显著,与对照组比较,差异均有统计学意义(P＜0.05)。
5.与健康组比较,两MG组AchRab水平显著增高,差异有统计学意义(P＜0.01)。两MG组IgA、IgM水平均显著低于健康组,差异有统计学意义(P＜0.01)。两MG组C_3、C_4水平显著降低,与健康组比较差异有统计学意义(P＜0.05)。经治疗后,治疗组补体C_3、C_4水平与治疗前相比显著增高,差异均有统计学意义(P＜0.05)。治疗组AchRab水平与治疗前比较有明显下降,差异有统计学意义(P＜0.01)。治疗组补体C_3、C_4及AchRab水平与对照组治疗后比较,差异有统计学意义(P＜0.05)。
结论:
1.黄芪复方+基础西药能明显改善重症肌无力患者的临床症状和中医证候,在中医证候改善方面明显优于单独应用强的松治疗,而且无任何毒副作用。
2.黄芪复方可能通过抑制Th细胞增殖,促进T细胞活化,降低CD4~+/CD8~+,使重症肌无力患者体内T淋巴细胞亚群比例重新分布,纠正重症肌无力异常的免疫应答过程,最终抑制B细胞产生以乙酰胆碱受体抗体为主的自身抗体。
3.黄芪复方通过抑制MG患者Th1型细胞和Tc1型细胞的分化,减少Th1/Tc1型细胞因子如IFN-γ的分泌而起到抗炎和改变病程的作用。
4.经过黄芪复方治疗后,血清中IFN-γ、IL-4、TNF-α均有不同程度的降低,可以反映中药治疗MG的免疫效应。在免疫抑制方面,黄芪复方+免疫抑制剂强的松治疗组比单用免疫抑制剂强的松效果明显,说明黄芪复方对调节免疫功能是双向的、多靶点的,而强的松对病人的免疫功能是过度抑制的,这显示出中药制剂黄芪复方的整体调节特征和优势。
5.黄芪复方规范化治疗以后MG患者血清中的补体C3、C4明显增高提示对MG患者测定血清补体,有助于进行动态观察,并可作为判断病情严重程度(活动性)的客观指标。
Myasthenia gravis is a kind of autoimmune disease,it mainly involves the Acetylcholine receptor(AchR) on the postsynaptic membrane of neuromuscular junction,mediated by the specific nicotinic acetylcholine receptor(N_2AchRab), and the complement participates its process.Its characteristics involve:1.Clinical symptoms:weakness of skeletat muscle,aggravated after action,and relieved after rest,slight in daytime and gravis at night.2.Electromyography:the amplitude decreased after echo-low frequency transduction of electric stimulation,at the same time,the miniature end-plate potential decreased,and the jitter of single fiber EMG widened,even blocked.3.Pharmacology:treated with cholinesterase inhibitor is effective, and over anaphylaxis to tubocurarine.4.Immunology:the increasing of acetylcholine receptor antibody in blood serum.5,Immunopathology:the wrinkles of postsynaptic membrane at neuromuscular junction decreased,flattened,and the AchR on the postsynaptic membrane decreased,myasthenia gravis is a cosmopolitan stubborn disease,the western medical treatment has its side effect, therefore,develop the effective Chinese patent medicine is the urgent mission. the complex recipe of Astragalus membranaceus is a pure Chinese drugs pharmaceutics,developed by Pro.Zhang Jingsheng under the directions of PISHEN theory,and obtained manifest therapeutic effect on the clinical and experimental study till now.
Purpose:Observe the complex recipe of Astragalus membranaceus's clinical effect in treating Myasthenia graves,provide its experimental basement;Observe the influence of the complex recipe of Astragalus membranaceus to T lymphocyte、Tc1/Tc2、Th1/Th2、IFN-γ、IL-4、TNF-α、IgG、IgA、IgM、C3、C4、AchRAb in peripheral blood of myasthenia gravis patients,reveal its immunological regulation effect on the cellulax level.
Methods:
1.According to the hospitalization date and the random-double blind-control principle,the myasthenia gravis patients was randomly divided into 2 groups, the curing group and the control group,30 cases per group.Patients of curing group administered the complex recipe of Astragalus membranaceus and prednisone placebo,Patients of control group administered prednisone tablet and Zhongjiling tablet placebo.Course of treatment is 90 days.Compare the pre-treatment and post-treatment clinical curative effect scores and Chinese medicals symptom scores.
2.Detect the pre-treatment and post-treatment distribution of T lymphocyte subgroup of myasthenia gravis patients with flow cytometry.
3.Detect the pre-treatment and post-treatment distribution of T lymphocyte subgroup Th1、Tc1、Th2、Tc2 of myasthenia gravis patients with flow cytometry.
4.Detect the the pre-treatment and post-treatment level of IFN-γ、IL-4、TNF-αof myasthenia gravis patients with ELISA assay technology。
5.Using the immunoturbidimetric and ELISA assay technology to detect the level of IgG、IgA、IgM、C3、C4、AChRAb in patients' peripheral blood pre-and post-administration.
Results:
1.Clinical research:The complex recipe of Astragalus membranaceus has significant effect to myasthenia gravis,the clinical syndrome score statistic result shows that the effective ratio of curing group is 70.0%,and that if control group is 56.7%,manifest difference exit between these two groups, P＜0.05;the change of Chinese medical symptom score statistic result shows that the effective ratio of curing group is 73.33%,and that if control group is 56.67%,manifest difference exit between these two groups,P＜0.01,it indicate that the curing group is better than that control group in improving Chinese medical symptoms.In this study,the patients in control group appeared side-effects such as sight hepatic dysfunction,increasing fasting serum glucose,femoral head necrosis and central obesity,on the contrary,The complex recipe of Astragalus membranaceus has exact curing effect without any side-effect,these shows that it is better than control group.
2.Before treatment,the CD4~+ cell percentage and CD4~+/CD8~+ of these two groups are higher than that of normal group,P＜0.01,the CD8~+ and NK(CD16~+/56~+)cell percentage of these two groups are lower than that of normal group,P＜0.01.The CD3~+ and B(CD19~+) cell percentage of these two groups have no difference with that of normal group,P＞0.05.After treatment,there is no change on the CD3~+ cell percentage of these two groups,P＞0.05,CD4~+ and CD19~+ cell percentage and the ratio of CD4~+/CD8~+ decreased significantly,CD8~+ and NK(CD16~+/56~+) cell increased significantly,P＜0.05.Compare with that of control group,there is significant difference for the CD4~+,CD8~+,CD4~+/CD8~+,CD19~+ and CD16~+/56~+ cell percentage,P＜0.05.
3.The Th1,Th2,Tc1 and Tc2 cell percentage of these two groups are higher than that of normal group,P＜0.01,Th1/Th2 and Tc1/Tc2 have difference with that of normal group,P＜0.05.After treatment,The Th1 and Tc1 cell percentage of the curing group decreased significantly,P＜0.01,there is obvious difference with that of control group.
4.The IFN-γ,IL-4 and TNF-αlevel of these two groups are significant higher than that of normal group,P＜0.01.After treatment,the IFN-γ,IL-4 and TNF-αlevel of two groups depressed,compared with that of pre-treatment, significant difference exited,P＜0.01.While in control group,the IFN-γ,IL-4 and TNF-αlevel decreased significantly,there is obvious difference with that of control group,P＜0.05.
5.The AchRab level of these two groups are higher than that of normal group, P＜0.01.The IgA and IgM level of these two groups are lower than that of normal group,P＜0.01.The C_3 and C_4 level of these two groups decreased significantly, there is obvious difference with that of normal group,P＜0.05.After treatment, the C_3 and C_4 level of curing group increased,compared with that of pre-treatment,significant difference exited,P＜0.05,the AChRAb level of the curing group decreased significantly,P＜0.01,while in control group,the C_3, C_4 and AchRab level are obvious difference with that of control group,P＜0.05.
Conclusion:
1.The complex recipe of Astragalus membranaceus can significantly improve the myasthenia gravis pationts' clinical symptoms and Chinese medicine syndromes,Its effect of improving Chinese medicine symptoms is better than that of only using prednisone,and no adverse reaction exited in this study.
2.The complex recipe of Astragalus membranaceus caused the redistribution of T lymphocyte subgroup ratio,this effect maybe due to its inhibition of Th cell proliferation,promoting Ts cell's activation and depress CD4~+/CD8~+ ratio, then rectified the abnormal immune response of myasthenia gravis patients, as a result,inhibited the production of auto antibody,mainly AchRab.
3.Th1 and Tc1 subsets decreased in the peripheral blood after MG patients were treated with leflunomide.The effect of leflunomide might be related to the prevention of Th1 and Tc1 differentiation and the decrease of Th1 and Tc1 cytokines such as IFN-γ.
4.After treatment with the complex recipe of Astragalus membranaceus,the IFN-γ, IL-4 and TNF-αlevel depressed,compared with that of pre-treatment,this indicated its immunological regulation effect.The complex recipe of Astragalus membranaceus+ prednisone has exact curing effect,it is better than that of only using prednisone.These indicate that The complex recipe of Astragalus membranaceus's immunological regulation is multi-targets and tow-way regulation.It is different with that of western medicine,which is overall inhibition to patients' immune function,and this manifest The complex recipe of Astragalus membranaceus's holo-regulation characteristic and predominance.
5.After treatment,the C_3 and C_4 level of curing group increased,it suggests that defecting of the levels of the complements in MG patients has attributions to mobile MG and maybe an objective index in determing the disease severity.
引文
[1]史玉泉主编.实用神经病学.上海:上海科学技术出版社,1994:880-881.
[2]卜碧涛,杨明山,徐金枝,等.长期使用泼尼松治疗重症肌无力有效性及前瞻性研究.中华神经科杂志.2000,33(1):28-31.
[3]许贤豪主编.神经免疫学.北京:北京医科大学中国协和医科大学联合出版社,2001:100-155.
[4]许贤豪主编.肌无力.北京:中国协和医科大学出版社,2003:48-75.
[5]周文泉.延缓衰老中药的筛选规程和临床观察规范.中西医结合杂志.1986,6(11):682.
[6]孙传兴主编.临床疾病诊断依据治愈好转标准.北京:人民军医出版社,1998:174-175.
[7]John TR.Long-term corticostteroid treatment of myastheniagravis.Ann N Y Acad Sci,1987,505:568 583.
[8]江明性.药理学.第3版.北京:人民卫生出版社,1990.284-290.
[9]徐冬波,蒋方建,李庚和,等.强力方对实验性自身免疫性重症肌无力细胞因子含量的影响.上海中医药杂志.2000,10:40.
[10]方沂主编.现代内科学[M].北京:人民军医出版社,1995.3702.
[11]潘邓记,杨明山,蔡玉祥.中西医结合治疗重症肌无力的临床观察.中国中西医结合杂志,2001,21(1):64.
[12]颧珠主编.实用内科学[M].北京:人民卫生出版社,1997.2236.
[13]李仪奎,姜名瑛主编.中药药理学[M].北京:中国中医药社,1992.186.
[14]许凤全,高学东.中西医结合治疗肌无力危象42例.河北中医.1998,4(20):235-236.
[15]许贤豪主编.神经免疫学.武汉:湖北科学技术出版社,2000:145-146.
[16]张广生.重症肌无力.中国实用乡村医生杂志.2007,14(2):6-8.
[17]Peritt D,Robertson S,Gri G,et al.Diferentiation of human NK cells into NK1and NK2 subsets.J Immunol.1998 Dec 1;161(11):5821-5824.
[18]郭振华,刘桂芳,高俊书,等.急性脑梗死CD3~+、CD4~+、CD8~+、CD4~+/CD8~+变化及其意义的研究.脑与神经疾病杂志.2005,13(3):223-224.
[19]潘利伟,郭蕾,林巍,等.重症急性胰腺炎患者CD4~+/CD8~+细胞比值、IL-16的变化及 意义.. 山东医药. 2005,45(28):54-55.
[20] Fambrough DM, Drachman DB, Satyamurti S. Neuromuscular junction in myasthenia gravis: decreased acetylcholine receptors. Science. 1973, 182:293-295.
[21] Almon KR, Andrew CG, Appel SH. Serum globulin in myasthenia gravis:inhibition of alpha-bungarotoxin binding to acetylcholine receptors. Science. 1974, 186:55-57.
[22] Lindsdrom JM, Lambert EH. Content of acetylcholine receptor in myasthenia gravis, experimental autoimmune myasthenia gravis and Eaton-Lambert syndrome.Neurology(NY).1978, 28:130-138.
[23] Lindstrom JM, Seybold ME, Lennon A. Antibody to acetylcholine receptor in myasthenia gravis: prevalence, clinical correlates and diagnostic value. Neurology(Minneap). 1976, 26:1054-1059.
[24] Mossman S, Vicent A, Newsom-Davis J. Myasthenia gravis without acetylcholine receptor antibody:a distinct disease entity. Lancet. 1986,1116-1119.
[25] Soliven BC, Lange DJ, Penn AS, et al. Seronegative myasthenia gravis.Neurology. 1988, 38:514-517.
[26] Drachman DB, Desilva S, Ramsay D, Pestronk A. "Sero-negative" myasthenia gravis:a humorally mediated variant of myasthenia. Neurology.1987 ,37:supl 1:214. abstract.
[27] Zhang GX, Xiao BG Bakhiet M, et al. Both CD4~+ and CD8~+ T cells are essential to induce experimental autoimmune myasthenia gravis. J Exp Med. 1996, Aug 1;184(2):349-356.
[28] Lisak RP, Abdou NI, Zweiman B, et al.Aspects of lymphocyte function in myasthenia gravis. Ann NY Acad Sci. 1976, 274:402-410.
[29] Lisak RP, Smiley R , Schotland DH, et al . Abnormalties of T-cell subpopulations in the blood and thymus of patients with myasthenia gravis. J Neurol Sci. 1979, 44:69-76.
[30] Piantelli M, Lauriola L, Carbone A, et al. Subpopulations of T lymphocytes in myasthenia gravis patients.Clin Exp Immunol. 1979, 36:85-89.
[31] A Melms, G Malcherek, U Cem, et al. Thymectomy and azathioprine have no effect on the phenotype of CD4 T lymphocyte subsets in myasthenia graivs. J Neurol Neurosurg Psychiatry. 1993, 56:46-51.
[32] Skolnik PR, Lisak RP, Zweiman B. Monoclonal antibody analysis of blood T-cell subsets in myasthenia gravis. Ann Neurol. 1982, 11:170-176.
[33] Matsui M, Fukuyama H, Akiguchi L, et al. Circulating CD4~+CD8~+ cells in myasthenia gravis:supplementary immunological parameter for long-term prognosis.J Neurol.1989, 236:329-335.
[34] Francesca Crosti,Marta Armanini, Paolo Confalotmieri, et al.Changes in peripheral blood lymphocyte subset frequencies in myasthenia gravis patients are related to immunosuppression. J Neurol. 1994, 241:218-222.
[35] I. Schlesinger, R . Rabinowitz, T. Brenner, et al. Changes in lymphocyte subsets in myasthenia gravis:Correlation with level of antibodies to acetylcholine receptor and age of patient. Neurology. 1992, 42:2153-2157.
[36] Andrea EM, James Hudson, Richard SA, et al. Immune regulation in myasthenia gra vis: Evidence for an increased suppressor T-cell population. Ann Neurol. 1982, 12:341-347.
[37] Berrich S, et al.Depletion of helper/inducer T cells after thymectomy in myasthenia gravis patients. Clin Immunol Immunopathol. 1983, 28:272-281.
[38] Shimizu H, IchikawaY, Yoshida M, et al. Lymphocyte subsets of the peripheral blood in myasthenia gravis determined by two-color flow cytometry. Autoimmunity. 1990, 6(3):173-182.
[39] Haynes BF, Harden EA, Olanow CW, et al.Effect of thymectomy on peripheral lymphocyte subsets in myasthenia gravis:selective effect on T-cells in patients with thymic atrophy. J Immunol. 1983, Aug;131(2):773-777.
[40] Fujii N, Itoyama Y, Tabira T, Kuroiwa Y. Fjiii N et al, Subsets of lymphoid cells in blood and thymus in myasthenia gravis. Monoclonal antibody analysis. J Neuroimmunol. 1983, Jun;4(3):151-159.
[41] Kawanami S, Mori S , Kamei H, et al.Cellular immunity in myasthenia gravis after thymectomy and corticosteroid therapy. Jpn J Med. 1990, Mar-Apr;29(2):138-145.
[42] Andrea EM, James Hudson, Richard SA, et al. Immune regulationin myasthenia gravis: Evidence for an increased suppressor T-cell population. Ann Neurol.1982,12:341-347.
[43] Chander Raman. CD5, an important regulator of lymphocyte selection and immune tolerance. Immunol Research. 2002, 26:255-263.
[44] Regheb S, Lisak RP. Phenotypic and functional abnormalities of B cells in myasthenia gravis. Ann N Y Acad Sci. 1993,Jun 21;681:256-263.
[45] 韩钊等.得症肌无力患者CD5~+B细胞的变化.中国免疫学杂志.2000,16:160-162.
[46] Mosmann TR, Cherwinski H, Bond MW, et al. Two types of murine Helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 1986, 136:2348.
[47] Romagnani S.Human Th1 and Th2 subsets:doubt no more.Immunol Today. 1991, 12:256.
[48] Mosmann TR, Schumacher JH, Street NF, et al. Diversity of cytokine synthesis and function of mouse CD4+T cells. Immunol Rev. 1991, 123:209.
[49] Woodland DL,Dutton RW. Heterogeneity of CD4~+ and CD8~+T cells.Curr Opin Immunol. 2003,15(3):336-342.
[50] Mosmann TR, Sad S. The expanding universe of T-cells ubsets:Th1, Th2 and more. Immunol Today. 1996,17(3):138-146.
[51] Jung T, Schauer U, Heusser C, et al.Detection of intracellular cytokines by flow cytometry.J Immunol Methods. 1993, 159:197.
[52] Maino VC , Piker LJ. Identification of functional subsets by flow cytometry:intracellular detection of cytokine expression.Cytometry. 1998, 34:207.
[53] Maecker HT, Maino WC, Pieker LJ. Immunofluoreseence analysis of T-cell responses in health and disease. J Clin Immunol. 2000, 20:391-399.
[54] Carter LL, Swain SL. Single cell analyses of cytokine produetion. Curr Opin Immunol. 1997, 9:177.
[55]胡大伟,石学耕,陈顺乐等.三色流式细胞术检测胞内细胞因子的建立.上海免疫学杂志.1999,19:250.
[56]Carter LL,Swain SL.Single cell analyses of cytokine Production.Curr Opin Immunol.1997,9:177.
[57]North ME,Ivory K,Funauchi M,et al.Intracellular cytokine production by human CO4~+ and CD8~+T cells from normal and immunodifieient donors using directly conjugated anti-cytokine antibodies and three-color flow cytometry.Clin Esp Immunol.1996,105:517.
[58]Nakano S.Engel AG Myasthenia gravis:quantitimmunocyic chemical analysis of inflammatory cells and detection of complement membrane attack complex at the end plate in 30 patients.Neurology.1993,13:1167-1178.
[59]LinK J,Soderstrom M,Jungdagl AL.Organ specific autoantigens induce intrefeon and Interleukin mRNA expression in mononuclear cells in multiple sclerosis and,myasthenia gravis.Neurology.1994,44:728-734.
[60]徐浩鹏,孙曼霁,周廷冲,丁氏双鳍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.1984,16(1):50-55.
[61]Stegall T,Krolick KA.Myocytes respond to both inter leukin-4 and interferon-gamma:cytokine respon-siveness with the potential to influence the severity and course of experimental myasthenia gravis.Clin Immunol.2000,94(2):133.
[62]Link H,Xiao BG.Rat models as tool to develop new immunotherapies.Immunol Rev.2001,184:117.
[63]Ma CG,Zhang GX,Xiao BG,et al.Cellular mRNA experimental of IFN-γ,IL-4 and transforming growth TGF-β in rat nasally tolerized.Clin Exp Immunol.1996,104(3):509-516.
[64]Ostlie N,Milani M,Wang W,et al.Absence of IL-4 facilitates the development of chyonic autoimmune myasthenia gravis in C57BL/6 mice.J Immunol.2003,170(1):604-612.
[65]Boom WH,Liano D,Abbas AK,et al.Heterogeneity of helper/inducer T Lymphocytes.Ⅱ.Effects of interleuking4 and interleukin2-producing T cell clones on resting B Lymphocytes.J Exp Med.1988,167(4):1350-1363.
[66]Zhong GX,Vaidirius Navikas,Hans Link.Cytokines and pathogenesis of myasthenia gravis.Muscle & Nerve.1997,20(5):543-551.
[67]Wang H B,Shi FD,Li HL,et al.Role for interferon-γ in rat strains with different susceptibility to experimental autoimmune myasthenia gravis.Clin Immunol.2000,95(2):156.
[68]Reyes-Reyna SM,Krolick KA.Chemokine production by rat myocytes exposed to interferon-gamma.Clin Immunol.2000,94(2):105.
[69]王炜,南振鸿,凌振芬,等.干扰素-γ对重症肌无力患者自身抗体的调节作用及相关性分析.中国神经免疫学和神经病学杂志.2000,7(4):234.
[70]Poea-Guyon S,Christadoss P,Panse RL,et al.Effects of cytokines on acetylcholine receptor expression:implications for myasthenia gravis.J Immunol.2005,174:5941.
[71]Goluszko E,Deng C,Possin MA,et al.Tumor necrosis receptor p55 and p75deficiency protects mice from developing experimental autoimmune myasthenia gravis.J Neuroimmunol.2002,122(1-2):85.
[72]姜广建,马立人,王润田,等.重症肌无力患者T细胞亚群和TNF-α检测及临床意义.临床荟萃.2002,17(21):246.
[73]胡军,阮丽荣,张清勇,等.重症肌无力患者外周血单个核细胞Fas及血清sFas、TNF检测.郑州大学学报(医学版).2005,40(1):4-6.
[74]闰晓波,王维治,韩威.重症肌无力cICAM-1及TNF-α水平的检测.中国神经免疫学和神经病学杂志.2002,9(2):80.
[75]Huang DR,Priskanen R,Matell G,et al.Tumor necrosis factor-alpha polymorphism and secretion in myasthenia gravis.Neuroimmunol.1999,94(1):165-171.
[76]赖祥青,杨明山,徐金枝,等.重症肌无力患者免疫发病机理的研究.中国病理生理学杂志.2001,17(7):662.
[77]Lindstrom J,et al.Myasthenia gravis.Adv-Immunol.1988,42:233-284.
[78]莫雪安等.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(2):20-23.
[79]高波廷.重症肌无力与三大自身抗体.国外医学神经病学神经外科学分册.1995,22(4):188-191.
[80]陈新谦,金有豫.新编药物学,第13版.北京:人民卫生出版社.1994,64-62.
[81]崔丽英,汤晓芙,许贤豪,等.重症肌无力的临床分型、电诊断及乙酞胆碱受体的比较.临床实用神经疾病杂志.1994,20:65.
[82]齐玲玲.重症肌无力的中医治疗.江西中医药.1998,29(3):17.
[83]Tindall RSA.Humoral immunityin myastheniagravis bio-chemical characteriaztion of acquiredanti-receptor antibody iesandclinical correlation.AnnNeurol.1981,(10):437.
[84]郑金瓯,莫雪安,马朝柱.重症肌无力IgG型及IgM型抗突触前膜抗体检测.卒中与神经疾病.1996,(3):185.
[85]乔健,李继民,肖保国.重症肌无力病人血清中IgM型乙酰胆碱受体、突触前膜抗体检测及临床意义.临床神经病学杂志.1998,11(2):87-89.
[86]Omi F.skeie Go,Vedeler C.et al.Complement activation by tit in and rganodine receptor autoantibodies in myasthenia gravis,A study of IgG subclasses and clinical correlations.J Neuroimmunol.2000,111(1-2):169-175.
[87]Lang TJ.Badea TC.Wade R.et al.Sublytic terminal complement at tack on myotubes decreases the expresssion of mRNAs encoding mus cle-specific proteins.J Neurochem.1997,Apr;68(4):1581-1589.
[88]Nakanos,Engel AG.Myasthenia gravis:Quantitative.immunocy-to-chemical analysis of inflammatory cellsand detection of complement membrane attach complex at the end-plate in 30 patients.Neurology.1993,43(6):1167-1172.
[89]Basta M,IIIa I.Dalakas MC.Increased in vitrouptake of the complement C3b in the serum of patients with guillain-Barre syndrome,myasthenia gravis and dermatomyositis.J Neuroimmunol,1996,71(1-2):227-229.
[90]Piddlesden SJ,Jiang S,Levin JL,vincent A,Morgan BP.Soluble comelement receetorl(sCR1)protects against exeerimental autoimmune myasthenia gravis.J Neuroimmunol.1996,71(1-2):173-177.
[91]余华葵、吴慧君、徐文祯.重症肌无力病人血清中补体成分的变化.华西医科大学学报.1991,22(4):438.
[92]蒋建明,管阳太.重症肌无力的研究历程与现状.中国神经科学杂志.2002,1:473.
[93]王云甫,孙圣刚.重症肌无力的研究现状.临床内科杂志.2004,1:168.
[94]魏雪飞,许贤豪,张华,等.重症肌无力患者胸腺细胞经AchR刺激后T淋巴细胞亚群Bcl-2的表达.中国神经免疫学和神经病学杂志.2006,13(1):1.
[95]李智伟,卓孝福.乙酰胆碱受体抗体量与眼型重症肌无力病情关系的实验观察.临床眼科杂志.2003,11(4):320.
[96]Lindstrom J M.Acetylcholine receptors and myasthenia.Muscle Never.2000,23(4):453.
[97]De DatesM,Stassen MH.Therole of antibodyes inmyasthenia gravis.Neuro Sci.2002,202(12):511.
[98]YangH,Wu B,T(u丨¨)z(u丨¨)n E,et al.Anewmouse model of autoimmune ocular myasthenia gravis.Invest Ophthalmol Vis Sci.2007,48(11):5101.
[99]B PMorgan,J Chamberlain-Banoub,J WNeal,et al.The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice.Clin Exp Immunol.2006,146(2):294.
[100]Ferdrik Romi,EinarK.Kristoffersen,Johan A.Aarli,Nils Erik Gilhus.The role of complement in myasthenia gravis:serological evidence of complement consumption in vivo.Jneuorimmunol.2005,158:191.
[101]Kmainski H,LI Z,Richmonds C.Complement regulators in extraocular musele and experimental autoimmune myasthenia gravis.Exp Neuorl.2004,189:333.
[102]蒋建明,管阳太.重症肌无力的研究历程与现状.中国神经科学杂志.2002,1:473.
[103]Ferdrik Romi,EinarK.Kristoffersen,Johan A.Aarli,Nils Erik Gilhus.The role of complement in myasthenia gravis:serological evidence of complement consumption in vivo.Jneuorimmunol.2005,158:191.
[104]王云甫,孙圣刚.重症肌无力的研究现状.临床内科杂志.2004,1:168.
[2]卜碧涛,杨明山,徐金枝,等.长期使用泼尼松治疗重症肌无力有效性及前瞻性研究.中华神经科杂志.2000,33(1):28-31.
[3]许贤豪主编.神经免疫学.北京:北京医科大学中国协和医科大学联合出版社,2001:100-155.
[4]许贤豪主编.肌无力.北京:中国协和医科大学出版社,2003:48-75.
[5]周文泉.延缓衰老中药的筛选规程和临床观察规范.中西医结合杂志.1986,6(11):682.
[6]孙传兴主编.临床疾病诊断依据治愈好转标准.北京:人民军医出版社,1998:174-175.
[7]John TR.Long-term corticostteroid treatment of myastheniagravis.Ann N Y Acad Sci,1987,505:568 583.
[8]江明性.药理学.第3版.北京:人民卫生出版社,1990.284-290.
[9]徐冬波,蒋方建,李庚和,等.强力方对实验性自身免疫性重症肌无力细胞因子含量的影响.上海中医药杂志.2000,10:40.
[10]方沂主编.现代内科学[M].北京:人民军医出版社,1995.3702.
[11]潘邓记,杨明山,蔡玉祥.中西医结合治疗重症肌无力的临床观察.中国中西医结合杂志,2001,21(1):64.
[12]颧珠主编.实用内科学[M].北京:人民卫生出版社,1997.2236.
[13]李仪奎,姜名瑛主编.中药药理学[M].北京:中国中医药社,1992.186.
[14]许凤全,高学东.中西医结合治疗肌无力危象42例.河北中医.1998,4(20):235-236.
[15]许贤豪主编.神经免疫学.武汉:湖北科学技术出版社,2000:145-146.
[16]张广生.重症肌无力.中国实用乡村医生杂志.2007,14(2):6-8.
[17]Peritt D,Robertson S,Gri G,et al.Diferentiation of human NK cells into NK1and NK2 subsets.J Immunol.1998 Dec 1;161(11):5821-5824.
[18]郭振华,刘桂芳,高俊书,等.急性脑梗死CD3~+、CD4~+、CD8~+、CD4~+/CD8~+变化及其意义的研究.脑与神经疾病杂志.2005,13(3):223-224.
[19]潘利伟,郭蕾,林巍,等.重症急性胰腺炎患者CD4~+/CD8~+细胞比值、IL-16的变化及 意义.. 山东医药. 2005,45(28):54-55.
[20] Fambrough DM, Drachman DB, Satyamurti S. Neuromuscular junction in myasthenia gravis: decreased acetylcholine receptors. Science. 1973, 182:293-295.
[21] Almon KR, Andrew CG, Appel SH. Serum globulin in myasthenia gravis:inhibition of alpha-bungarotoxin binding to acetylcholine receptors. Science. 1974, 186:55-57.
[22] Lindsdrom JM, Lambert EH. Content of acetylcholine receptor in myasthenia gravis, experimental autoimmune myasthenia gravis and Eaton-Lambert syndrome.Neurology(NY).1978, 28:130-138.
[23] Lindstrom JM, Seybold ME, Lennon A. Antibody to acetylcholine receptor in myasthenia gravis: prevalence, clinical correlates and diagnostic value. Neurology(Minneap). 1976, 26:1054-1059.
[24] Mossman S, Vicent A, Newsom-Davis J. Myasthenia gravis without acetylcholine receptor antibody:a distinct disease entity. Lancet. 1986,1116-1119.
[25] Soliven BC, Lange DJ, Penn AS, et al. Seronegative myasthenia gravis.Neurology. 1988, 38:514-517.
[26] Drachman DB, Desilva S, Ramsay D, Pestronk A. "Sero-negative" myasthenia gravis:a humorally mediated variant of myasthenia. Neurology.1987 ,37:supl 1:214. abstract.
[27] Zhang GX, Xiao BG Bakhiet M, et al. Both CD4~+ and CD8~+ T cells are essential to induce experimental autoimmune myasthenia gravis. J Exp Med. 1996, Aug 1;184(2):349-356.
[28] Lisak RP, Abdou NI, Zweiman B, et al.Aspects of lymphocyte function in myasthenia gravis. Ann NY Acad Sci. 1976, 274:402-410.
[29] Lisak RP, Smiley R , Schotland DH, et al . Abnormalties of T-cell subpopulations in the blood and thymus of patients with myasthenia gravis. J Neurol Sci. 1979, 44:69-76.
[30] Piantelli M, Lauriola L, Carbone A, et al. Subpopulations of T lymphocytes in myasthenia gravis patients.Clin Exp Immunol. 1979, 36:85-89.
[31] A Melms, G Malcherek, U Cem, et al. Thymectomy and azathioprine have no effect on the phenotype of CD4 T lymphocyte subsets in myasthenia graivs. J Neurol Neurosurg Psychiatry. 1993, 56:46-51.
[32] Skolnik PR, Lisak RP, Zweiman B. Monoclonal antibody analysis of blood T-cell subsets in myasthenia gravis. Ann Neurol. 1982, 11:170-176.
[33] Matsui M, Fukuyama H, Akiguchi L, et al. Circulating CD4~+CD8~+ cells in myasthenia gravis:supplementary immunological parameter for long-term prognosis.J Neurol.1989, 236:329-335.
[34] Francesca Crosti,Marta Armanini, Paolo Confalotmieri, et al.Changes in peripheral blood lymphocyte subset frequencies in myasthenia gravis patients are related to immunosuppression. J Neurol. 1994, 241:218-222.
[35] I. Schlesinger, R . Rabinowitz, T. Brenner, et al. Changes in lymphocyte subsets in myasthenia gravis:Correlation with level of antibodies to acetylcholine receptor and age of patient. Neurology. 1992, 42:2153-2157.
[36] Andrea EM, James Hudson, Richard SA, et al. Immune regulation in myasthenia gra vis: Evidence for an increased suppressor T-cell population. Ann Neurol. 1982, 12:341-347.
[37] Berrich S, et al.Depletion of helper/inducer T cells after thymectomy in myasthenia gravis patients. Clin Immunol Immunopathol. 1983, 28:272-281.
[38] Shimizu H, IchikawaY, Yoshida M, et al. Lymphocyte subsets of the peripheral blood in myasthenia gravis determined by two-color flow cytometry. Autoimmunity. 1990, 6(3):173-182.
[39] Haynes BF, Harden EA, Olanow CW, et al.Effect of thymectomy on peripheral lymphocyte subsets in myasthenia gravis:selective effect on T-cells in patients with thymic atrophy. J Immunol. 1983, Aug;131(2):773-777.
[40] Fujii N, Itoyama Y, Tabira T, Kuroiwa Y. Fjiii N et al, Subsets of lymphoid cells in blood and thymus in myasthenia gravis. Monoclonal antibody analysis. J Neuroimmunol. 1983, Jun;4(3):151-159.
[41] Kawanami S, Mori S , Kamei H, et al.Cellular immunity in myasthenia gravis after thymectomy and corticosteroid therapy. Jpn J Med. 1990, Mar-Apr;29(2):138-145.
[42] Andrea EM, James Hudson, Richard SA, et al. Immune regulationin myasthenia gravis: Evidence for an increased suppressor T-cell population. Ann Neurol.1982,12:341-347.
[43] Chander Raman. CD5, an important regulator of lymphocyte selection and immune tolerance. Immunol Research. 2002, 26:255-263.
[44] Regheb S, Lisak RP. Phenotypic and functional abnormalities of B cells in myasthenia gravis. Ann N Y Acad Sci. 1993,Jun 21;681:256-263.
[45] 韩钊等.得症肌无力患者CD5~+B细胞的变化.中国免疫学杂志.2000,16:160-162.
[46] Mosmann TR, Cherwinski H, Bond MW, et al. Two types of murine Helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol. 1986, 136:2348.
[47] Romagnani S.Human Th1 and Th2 subsets:doubt no more.Immunol Today. 1991, 12:256.
[48] Mosmann TR, Schumacher JH, Street NF, et al. Diversity of cytokine synthesis and function of mouse CD4+T cells. Immunol Rev. 1991, 123:209.
[49] Woodland DL,Dutton RW. Heterogeneity of CD4~+ and CD8~+T cells.Curr Opin Immunol. 2003,15(3):336-342.
[50] Mosmann TR, Sad S. The expanding universe of T-cells ubsets:Th1, Th2 and more. Immunol Today. 1996,17(3):138-146.
[51] Jung T, Schauer U, Heusser C, et al.Detection of intracellular cytokines by flow cytometry.J Immunol Methods. 1993, 159:197.
[52] Maino VC , Piker LJ. Identification of functional subsets by flow cytometry:intracellular detection of cytokine expression.Cytometry. 1998, 34:207.
[53] Maecker HT, Maino WC, Pieker LJ. Immunofluoreseence analysis of T-cell responses in health and disease. J Clin Immunol. 2000, 20:391-399.
[54] Carter LL, Swain SL. Single cell analyses of cytokine produetion. Curr Opin Immunol. 1997, 9:177.
[55]胡大伟,石学耕,陈顺乐等.三色流式细胞术检测胞内细胞因子的建立.上海免疫学杂志.1999,19:250.
[56]Carter LL,Swain SL.Single cell analyses of cytokine Production.Curr Opin Immunol.1997,9:177.
[57]North ME,Ivory K,Funauchi M,et al.Intracellular cytokine production by human CO4~+ and CD8~+T cells from normal and immunodifieient donors using directly conjugated anti-cytokine antibodies and three-color flow cytometry.Clin Esp Immunol.1996,105:517.
[58]Nakano S.Engel AG Myasthenia gravis:quantitimmunocyic chemical analysis of inflammatory cells and detection of complement membrane attack complex at the end plate in 30 patients.Neurology.1993,13:1167-1178.
[59]LinK J,Soderstrom M,Jungdagl AL.Organ specific autoantigens induce intrefeon and Interleukin mRNA expression in mononuclear cells in multiple sclerosis and,myasthenia gravis.Neurology.1994,44:728-734.
[60]徐浩鹏,孙曼霁,周廷冲,丁氏双鳍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.1984,16(1):50-55.
[61]Stegall T,Krolick KA.Myocytes respond to both inter leukin-4 and interferon-gamma:cytokine respon-siveness with the potential to influence the severity and course of experimental myasthenia gravis.Clin Immunol.2000,94(2):133.
[62]Link H,Xiao BG.Rat models as tool to develop new immunotherapies.Immunol Rev.2001,184:117.
[63]Ma CG,Zhang GX,Xiao BG,et al.Cellular mRNA experimental of IFN-γ,IL-4 and transforming growth TGF-β in rat nasally tolerized.Clin Exp Immunol.1996,104(3):509-516.
[64]Ostlie N,Milani M,Wang W,et al.Absence of IL-4 facilitates the development of chyonic autoimmune myasthenia gravis in C57BL/6 mice.J Immunol.2003,170(1):604-612.
[65]Boom WH,Liano D,Abbas AK,et al.Heterogeneity of helper/inducer T Lymphocytes.Ⅱ.Effects of interleuking4 and interleukin2-producing T cell clones on resting B Lymphocytes.J Exp Med.1988,167(4):1350-1363.
[66]Zhong GX,Vaidirius Navikas,Hans Link.Cytokines and pathogenesis of myasthenia gravis.Muscle & Nerve.1997,20(5):543-551.
[67]Wang H B,Shi FD,Li HL,et al.Role for interferon-γ in rat strains with different susceptibility to experimental autoimmune myasthenia gravis.Clin Immunol.2000,95(2):156.
[68]Reyes-Reyna SM,Krolick KA.Chemokine production by rat myocytes exposed to interferon-gamma.Clin Immunol.2000,94(2):105.
[69]王炜,南振鸿,凌振芬,等.干扰素-γ对重症肌无力患者自身抗体的调节作用及相关性分析.中国神经免疫学和神经病学杂志.2000,7(4):234.
[70]Poea-Guyon S,Christadoss P,Panse RL,et al.Effects of cytokines on acetylcholine receptor expression:implications for myasthenia gravis.J Immunol.2005,174:5941.
[71]Goluszko E,Deng C,Possin MA,et al.Tumor necrosis receptor p55 and p75deficiency protects mice from developing experimental autoimmune myasthenia gravis.J Neuroimmunol.2002,122(1-2):85.
[72]姜广建,马立人,王润田,等.重症肌无力患者T细胞亚群和TNF-α检测及临床意义.临床荟萃.2002,17(21):246.
[73]胡军,阮丽荣,张清勇,等.重症肌无力患者外周血单个核细胞Fas及血清sFas、TNF检测.郑州大学学报(医学版).2005,40(1):4-6.
[74]闰晓波,王维治,韩威.重症肌无力cICAM-1及TNF-α水平的检测.中国神经免疫学和神经病学杂志.2002,9(2):80.
[75]Huang DR,Priskanen R,Matell G,et al.Tumor necrosis factor-alpha polymorphism and secretion in myasthenia gravis.Neuroimmunol.1999,94(1):165-171.
[76]赖祥青,杨明山,徐金枝,等.重症肌无力患者免疫发病机理的研究.中国病理生理学杂志.2001,17(7):662.
[77]Lindstrom J,et al.Myasthenia gravis.Adv-Immunol.1988,42:233-284.
[78]莫雪安等.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(2):20-23.
[79]高波廷.重症肌无力与三大自身抗体.国外医学神经病学神经外科学分册.1995,22(4):188-191.
[80]陈新谦,金有豫.新编药物学,第13版.北京:人民卫生出版社.1994,64-62.
[81]崔丽英,汤晓芙,许贤豪,等.重症肌无力的临床分型、电诊断及乙酞胆碱受体的比较.临床实用神经疾病杂志.1994,20:65.
[82]齐玲玲.重症肌无力的中医治疗.江西中医药.1998,29(3):17.
[83]Tindall RSA.Humoral immunityin myastheniagravis bio-chemical characteriaztion of acquiredanti-receptor antibody iesandclinical correlation.AnnNeurol.1981,(10):437.
[84]郑金瓯,莫雪安,马朝柱.重症肌无力IgG型及IgM型抗突触前膜抗体检测.卒中与神经疾病.1996,(3):185.
[85]乔健,李继民,肖保国.重症肌无力病人血清中IgM型乙酰胆碱受体、突触前膜抗体检测及临床意义.临床神经病学杂志.1998,11(2):87-89.
[86]Omi F.skeie Go,Vedeler C.et al.Complement activation by tit in and rganodine receptor autoantibodies in myasthenia gravis,A study of IgG subclasses and clinical correlations.J Neuroimmunol.2000,111(1-2):169-175.
[87]Lang TJ.Badea TC.Wade R.et al.Sublytic terminal complement at tack on myotubes decreases the expresssion of mRNAs encoding mus cle-specific proteins.J Neurochem.1997,Apr;68(4):1581-1589.
[88]Nakanos,Engel AG.Myasthenia gravis:Quantitative.immunocy-to-chemical analysis of inflammatory cellsand detection of complement membrane attach complex at the end-plate in 30 patients.Neurology.1993,43(6):1167-1172.
[89]Basta M,IIIa I.Dalakas MC.Increased in vitrouptake of the complement C3b in the serum of patients with guillain-Barre syndrome,myasthenia gravis and dermatomyositis.J Neuroimmunol,1996,71(1-2):227-229.
[90]Piddlesden SJ,Jiang S,Levin JL,vincent A,Morgan BP.Soluble comelement receetorl(sCR1)protects against exeerimental autoimmune myasthenia gravis.J Neuroimmunol.1996,71(1-2):173-177.
[91]余华葵、吴慧君、徐文祯.重症肌无力病人血清中补体成分的变化.华西医科大学学报.1991,22(4):438.
[92]蒋建明,管阳太.重症肌无力的研究历程与现状.中国神经科学杂志.2002,1:473.
[93]王云甫,孙圣刚.重症肌无力的研究现状.临床内科杂志.2004,1:168.
[94]魏雪飞,许贤豪,张华,等.重症肌无力患者胸腺细胞经AchR刺激后T淋巴细胞亚群Bcl-2的表达.中国神经免疫学和神经病学杂志.2006,13(1):1.
[95]李智伟,卓孝福.乙酰胆碱受体抗体量与眼型重症肌无力病情关系的实验观察.临床眼科杂志.2003,11(4):320.
[96]Lindstrom J M.Acetylcholine receptors and myasthenia.Muscle Never.2000,23(4):453.
[97]De DatesM,Stassen MH.Therole of antibodyes inmyasthenia gravis.Neuro Sci.2002,202(12):511.
[98]YangH,Wu B,T(u丨¨)z(u丨¨)n E,et al.Anewmouse model of autoimmune ocular myasthenia gravis.Invest Ophthalmol Vis Sci.2007,48(11):5101.
[99]B PMorgan,J Chamberlain-Banoub,J WNeal,et al.The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice.Clin Exp Immunol.2006,146(2):294.
[100]Ferdrik Romi,EinarK.Kristoffersen,Johan A.Aarli,Nils Erik Gilhus.The role of complement in myasthenia gravis:serological evidence of complement consumption in vivo.Jneuorimmunol.2005,158:191.
[101]Kmainski H,LI Z,Richmonds C.Complement regulators in extraocular musele and experimental autoimmune myasthenia gravis.Exp Neuorl.2004,189:333.
[102]蒋建明,管阳太.重症肌无力的研究历程与现状.中国神经科学杂志.2002,1:473.
[103]Ferdrik Romi,EinarK.Kristoffersen,Johan A.Aarli,Nils Erik Gilhus.The role of complement in myasthenia gravis:serological evidence of complement consumption in vivo.Jneuorimmunol.2005,158:191.
[104]王云甫,孙圣刚.重症肌无力的研究现状.临床内科杂志.2004,1:168.