实验性重症肌无力大鼠模型的研究

目的从丁(氏)双鳍电鳐的电器官分离乙酰胆碱受体(AChR)免疫大鼠,建立实验性自身免疫性重症肌无力(EAMG)动物模型.方法参照徐浩鹏等方法从丁(氏)双鳍电鳐的电器官提取AChR蛋白,并采用Folin-酚试剂法及酶联免疫吸附法(ELISA)检测提取蛋白的含量及活性;以提取的蛋白主动免疫Lewis大鼠,每天观察其临床表现,并于免疫后7周进行低频重复电刺激、单纤维肌电图及血清中乙酰胆碱受体抗体(AchRAb)水平检测.结果提取蛋白的含量为35.4 mg/mL,AChR提取物可与阳性血清中的AChRAb发生结合反应.免疫后1周左右,实验组有3只大鼠出现轻度肌无力症状,持续2～5 d自行缓解;免疫后5周左右实验组8只大鼠均表现出不同程度肌无力症状.实验组大鼠低频重复电刺激阳性率达75%,其衰减率明显高于对照组.实验组大鼠单肌纤维动作电位平均连续差(MCD)值与正常对照组比较明显延长(P＜0.01),而福(氏)完全佐剂(CFA)对照组与正常对照组间MCD值差异无显著性.实验组大鼠AChRAb水平明显高于CFA对照组(P＜0.01);实验组7只大鼠AChRAb的P/N值≥2.1,而CFA对昭组P/N值均＜1.4.结论从丁(氏)双鳍电鳐电器官提取的AChR蛋白成功地诱导成EAMG动物模型,可为进一步研究重症肌无力创造一定条件.

Study on Animal Model of Experimental Autoimmune Myasthenia Gravis in Rats

Objective To study the rats model of experimental autoimmune myasthenia gravis (EAMG) induced with extract from electric organ of narcine timilei. Methods According to Xu Hao-peng method, acetylcholine receptor (AChR) protein was extracted from electric organ of narcine timilei, and identified with Folin-phenol reagent and enzyme-linked immunoadsordent assay (ELISA). Lewis rats were immunized with the extracted protein and completed Freund adjuvant (CFA) in experiment group, and with CFA alone in CFA group. The clinical manifestations were observed every day. After 7 weeks, repetitive nerve stimulation (RNS), single fibre electromyography (SFEMG) and the levels of acetylcholine receptor antibody (AChRAb) were detected. Results The content of the protein was 35. 4 mg/mL,and the protein could respond with AChRAb from the serum of seropositive myasthenia gravis (MG) patient. The mild symptoms were observed on 3 experimental rats after a week, which lasted for 2 to 5 days. And after 5 weeks, all experimental rats presented some symptoms at different degree like MG. The positive rate of RNS of the experimental group was 75%, and the percentage of decrement of these rats was higher than that of control rats. The mean consecutived difference (MCD) of,experimental rats were longer than that of control rats markedly ( P <0. 01). The levels of AChRAb of experimental rats were higher than that of CFA control rats significantly ( P <0. 01), and the values of P/N of 7 experimental rats were more than 2. 1, but those of the control rats were less than 1. 4. Conclusions It is pointed out that the extract from electric organ of narcine timilei could serve as the antigen to induce EAMG in rats.