【作者】 李良龙；

【导师】 刘友章；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2009， 博士

【摘要】 重症肌无力(Myasthenia Gravis,MG)是人类的一种由针对神经肌肉接头突触后膜上乙酰胆碱受体(AChR)的自身抗体介导的器官特异自身免疫疾病,其临床表现主要有部分或骨骼肌易疲劳,呈波动性肌无力,具体活动后加重、休息后减轻和晨轻暮重等,约1%的MG患者可出现肌无力危象,抢救不及时,可引起死亡。健脾祛湿方是导师刘友章教授临床长期用于治疗重症肌无力的中药复方,临床治疗MG患者,效果明显。为探讨健脾祛湿方的免疫调节作用的机理,我们用皮下注射鼠源性AchR-α亚基97～116肽段方法,建立实验性自身免疫性重症肌无力模型。展开T细胞及其亚群及细胞因子的研究。目的:探讨健脾祛湿方对实验性自身免疫性重症肌无力大鼠的治疗机理。方法:用皮下注射鼠源性AchR-α亚基97～116肽段方法,建立实验性自身免疫性重症肌无力模型,针对MG的基本病机是脾气虚兼有湿热组成健脾祛湿方,用健脾祛湿方、强的松给大鼠灌胃4周,处死动物。利用流式细胞技术和免疫组织化学方法,分别以T细胞亚群为指标,观察健脾祛湿方、强的松对EAMG细胞免疫的影响;以IL-6、TGF-β1为指标,观察健脾祛湿方对EAMG细胞因子的影响。结果:模型评价:1.Lennon临床评分达0.5～1级,且随着时间的延长肌无力症状愈加显著,至造模结束时临床评分在1级以上者占造模鼠的78.38%。2.应用5Hz连续10次的低频电刺激检测36只造模实验鼠,共有32只大鼠第1与第5个电位的衰减百分比超过10%,平均为(20.42±4.45),其余4只大鼠的电衰减率小于10%,而对照组最大衰减均不超过10%中药干预后:模型组AchR-Ab含量明显增高,与正常组相比具有显著差异(P＜0.01);进行药物干预后,AchR-Ab含量均较模型组明显降低(P＜0.05),但健脾清热祛湿方低剂量组AchR-Ab含量均明显高于强的松组(P＜0.05)。健脾清热祛湿方高剂量组与西药组比较差异无统计学意义(P＞0.05)。模型组IL-6含量明显增高,与正常组相比差异有统计学意义(P＜0.05);进行药物干预后,IL-6含量均较模型组明显降低(P＜0.05),但健脾祛湿方低剂量组IL-6含量均明显高于强的松组(P＜0.05)。健脾祛湿方高剂量组与低剂量组比较差异无统计学意义(P＞0.05)。模型组TGF-β1含量明显降低,与正常组相比具有差异(P＜0.05);进行药物干预后,TGF-β1含量均较模型组明显增加(P＜0.05),但健脾祛湿方高低剂量组TGF-β1含量均低于强的松组(P＜0.05)。健脾祛湿方高剂量组与低剂量组比较差异无统计学意义(P＞0.05)。健脾祛湿方低剂量组与正常组比较差异无统计学意义。CD4~+%的比较:强的松组与模型组比较差异有统计学意义(P＜0.05);模型组与正常组相比具有差异无统计学意义(P＞0.05);健脾祛湿方高剂量组与低剂量组比较差异无统计学意义(P＞0.05)。正常组与健脾祛湿方高、低剂量组比较,差异有统计学意义(P＜0.05)。强的松组与健脾祛湿方高、低剂量组比较,差异有统计学意义(P＜0.05)CD8~+%的比较:正常组与强的松组比较差异有统计学意义(P＜0.05);强的松组与中药低剂量组比较差异有统计学意义(P＜0.05)。CD4~+/CD8~+的比较:正常组与健脾祛湿方高、低剂量组比较,差异有统计学意义(P＜0.05)。强的松组与健脾祛湿方高、低剂量组比较,差异有统计学意义(P＜0.05)结论:1、用皮下注射鼠源性AchR-α亚基97～116肽段可成功制作EAMG模型。2、健脾祛湿方能改善EAMG的肌无力症状。健脾祛湿方能升高EAMG大鼠CD4~+/CD8~+比值,强的松则减低比值。强的松、健脾祛湿方均能改善EAMG症状,降低AchR-Ab浓度值,降低IL-6水平,健脾祛湿方能使EAMG大鼠TGF-β1接近正常。强的松、健脾祛湿方治疗EAMG的机理是不一致的,强的松是通过免疫抑制来改善EAMG的肌无力症状,而健脾祛湿方则是通过提高免疫来改善肌无力症状的可见,健脾祛湿方通过影响T细胞和其亚群及细胞因子,调控细胞免疫和体液免疫,起到治疗MG的作用。更多还原

【Abstract】 Myasthenia gravis(MG) is a T cell-regulated,antibody-mediated autoimmune disease.Impaired neuromuscular transmission characteristic of MG results from antibodies against the nicotinic acetylcholine receptor(AchR) of skeletal muscle.Experimental autoimmune myasthenia gravis(EAMG)is a well-established animal model for human myasthenia gravis(MG).The IL-6, TGF-β1,CD4~+/CD8~+,might be involved in the developed of both MG and EAMG,while TGF-β1may have suppressive effect in both diseases.Jianpiqushi Prescription is an effective herbal prescription to treat Myasthenia gravis(MG).Jianpiqushi Prescription has been used to treat thousands of MG cases and got good result.This thesis,which establishing EAMG model in rats and focusing on cytokines IL-6,TGF-β1 etc,to studied the functional mechanism of Jianpiqushi Prescription preventing from MG.Objective:Studied the therapeutic mechanism of Jianpiqushi Prescription to EAMG.Method:To investigate the influence of Jianpiqushi Prescription to experimental autoimmune myasthenia gravis(EAMG).Lewis rats were immunized with Torpedo-derived 97-116 peptide of the AchRα-subunit in CFA.After the third immunization,the rats’ weights,symptoms,activities electromyograms were observed to judgement the EAMG Model were succeed made.Then these succeed made EAMG model were administered by Prednisone and Jianpiqushi Prescription,and killed after 4 weeks.By using the flowcytometric analysis and immunohistochemical technique,the effects of the Jianpiqushi Prescription and Prednisone on the T lymphocyte and T lymphocyte subgroups of the EAMG of rats were observed respectively.The effect of Jianpiqushi Prescription and Prednisone on the EAMG cytokines was observed by examining the index of IL-6, TGF-β1,AchR-Ab.Results:model judgement:There were 32 successful EAMG models of 39 Lewis rats.EAMG models were easy to fatigue.The weights of them reduced.The results of the percentage of decrement of RNS had significent differenc from normal rats.The results of the effect of Jianpiqushi Prescription and Prednisone on the EAMG were as below:Jianpiqushi PrescriPtion could improve clinical symptoms,increase body weight,increase the titers of TGF-β1 and decrease titers of AChR-Ab、IL-6,comparing from the model group.The AchR-Ab indexes were no significent differences between Jianpiqushi Prescription in high dosage group and Prednisone group.Compared with Prednisone grou,the levets of AchR-Ab of Jianpiqushi Prescription in low dosage group were heightened.Effect of Jianpiqushi Prescription on cytokine of EAMG rats: Compared with CFA group,levels of IL-6 of model group were heightened remarkably,P＜0.01.Compared with Prednisone grou,the levels of IL-6 of Jianpiqushi Prescription in low dosage group were heightened。The IL-6 indexes were no significent differences between Jianpiqushi Prescription in high dosage group and low dosage group.Compared with CFA group,levels of TGF-β1 of model group were declined. Compared with Prednisone grou,the levels of TGF-β1 of Jianpiqushi Prescription in high and low dosage group were both declined.The TGF-β1 indexes were no significent differences between Jianpiqushi Prescription in high dosage group and low dosage group.The ratio of CD4~+:Compared with Prednisone grou,the ratio of CD4~+ of model group were heightened.The ratio of CD4~+ were no significent differences between model group and CFA group.There were no significent differences between Jianpiqushi Prescription in high dosage group and low dosage group.The ratio of CD8~+: Compared with Prednisone group,the ratio of CD8~+ of model group were heightened.Compared with Prednisone group,the ratio of CD8~+ of Jianpiqushi Prescription in low dosage group were heightened.Compared with Jianpiqushi Prescription in high dosage group and low dosage group,the ratio CD4~+/CD8~+ of the CFA group was decreased significantly.Conclusions:Jianpiqushi Prescription can improve the lack strength of muscles and regulate the Tlymphocyte and antibody immunological function through impacting on the Tlymphocyte and Tlymphocyte subgroup and cell factor to treat MG.Jianpiqushi Prescription could increased the ratio of CD4~+/CD8~+,but Prednisone was decreased.Prednisone could more decreased the levels of IL-6 and AchR-Ab than the Jianpiqushi Prescription.Jianpiqushi Prescription could increased the levels of TGF-β1 to normal.更多还原