低密度脂蛋白受体相关蛋白4对小鼠重症肌无力的诱导

发布时间：2018-03-21 12:31

本文选题：低密度脂蛋白受体相关蛋白4(LRP4)　切入点：抗低密度脂蛋白受体相关蛋白4抗体　出处：《延边大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:探讨位于神经肌肉接头处(neuromuscular junction,NMJ)的低密度脂蛋白受体相关蛋白 4(low density lipoprotein receptor-related protein 4,LRP4)是否能诱导出小鼠重症肌无力(myasthenia gravis,MG),以证明抗LRP4抗体是否为MG的致病性抗体。方法:建立小鼠实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis,EAMG)动物模型。选取10只8周龄雌性C57BL/6小鼠,随机分为实验组和对照组。采用主动免疫的方法,第1天进行首次注射,将LRP4与完全弗氏佐剂(CFA)免疫实验组小鼠。每天观察小鼠的饮食饮水情况以及有无临床症状出现,每48小时称量1次小鼠的体重变化。首次免疫后,分别于第28天,第56天进行加强免疫。最后1次免疫后第7天处死两组小鼠,并取下小鼠腓肠肌,制作冰冻切片。切片用四甲基罗丹明结合的银环蛇毒素定位乙酰胆碱受体(acetylcholine receptor,AChR)(红色),用FITC结合的羊抗小鼠IgG抗体定位抗LRP4抗体(绿色),在激光共聚焦显微镜下观察。同时用透射电镜观察NMJ的超微结构变化。结果:实验组与对照组小鼠未出现抓握和嘶叫无力的明显临床症状。实验组1只小鼠出现眼睛无法睁开的临床表现。激光共聚焦显微镜观察后发现,实验组腓肠肌的细胞膜表面存在绿色荧光,同一位置存在红色荧光,表明抗LRP4抗体已经与抗原LRP4结合,而且结合部位为NMJ的AChR处。透射电镜观察实验组小鼠肌细胞发现,突触结构形态不规则,分支简化且分散,符合MG的病理变化特点。而对照组突触结构分支复杂且连续,神经末梢分支广泛。结论:抗LRP4抗体已经结合在NMJ突触后膜的AChR部位的抗原LRP4上,导致突触后膜出现超微结构病理改变,并引起小鼠眼肌无力的临床表现,初步证明LRP4可以诱导小鼠MG,抗LRP4抗体是MG的致病性抗体。
[Abstract]:Objective: to investigate whether the low density lipoprotein receptor-associated protein (4low density lipoprotein receptor-related protein 4LRP4) located at the neuromuscular junction (NMJ) can induce myasthenia graviscapus myasthenia gravis MGG in mice, so as to prove whether anti-#en4# antibody is the pathogenic antibody of MG. Methods: to establish the experimental autoimmune autoimmune myasthenia gravida (EAMG) animal model in mice. Ten 8-week-old female C57BL / 6 mice were selected. The mice in the experimental group were immunized with LRP4 and Freund's adjuvant on the first day. After the first immunization, the mice were immunized on the 28th day and the 56th day respectively. The mice were killed on the 7th day after the last immunization, and the gastrocnemius muscle was removed. The frozen sections were made. The sections were located with acetylcholine receptor acetylcholine receptor acetylcholine receptor acetylcholine receptor (ACHR) with tetramethyl rhodamine and IgG antibody (green) with FITC binding goat anti mouse IgG antibody (green) under laser confocal microscope. At the same time, the ultrastructural changes of NMJ were observed by transmission electron microscope. Results: the mice in the experimental group and the control group showed no obvious clinical symptoms of grasping and screaming weakness, and one mouse in the experimental group showed the clinical manifestation that the eyes could not be opened. Laser confocal microscopy showed that, There was green fluorescence on the membrane surface of gastrocnemius muscle in the experimental group, and red fluorescence in the same position, which indicated that the anti LRP4 antibody had been bound to the antigenic LRP4, and the binding site was at the AChR site of NMJ. The synaptic structure was irregular, the branches were simplified and scattered, which was consistent with the pathological changes of MG, while in the control group, the branches of synaptic structure were complex and continuous. Conclusion: the anti LRP4 antibody has been bound to the antigenic LRP4 of the AChR site of the postsynaptic membrane of NMJ, resulting in ultrastructural pathological changes of the postsynaptic membrane and the clinical manifestations of mouse ophthalmoasthenia. The results showed that LRP4 could induce MG. anti LRP4 antibody was the pathogenicity antibody of MG.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R746.1

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