本文選題：低密度脂蛋白受體相關(guān)蛋白4(LRP4)　切入點(diǎn)：抗低密度脂蛋白受體相關(guān)蛋白4抗體　出處：《延邊大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討位于神經(jīng)肌肉接頭處(neuromuscular junction,NMJ)的低密度脂蛋白受體相關(guān)蛋白 4(low density lipoprotein receptor-related protein 4,LRP4)是否能誘導(dǎo)出小鼠重癥肌無(wú)力(myasthenia gravis,MG),以證明抗LRP4抗體是否為MG的致病性抗體。方法:建立小鼠實(shí)驗(yàn)性自身免疫性重癥肌無(wú)力(experimental autoimmune myasthenia gravis,EAMG)動(dòng)物模型。選取10只8周齡雌性C57BL/6小鼠,隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組。采用主動(dòng)免疫的方法,第1天進(jìn)行首次注射,將LRP4與完全弗氏佐劑(CFA)免疫實(shí)驗(yàn)組小鼠。每天觀察小鼠的飲食飲水情況以及有無(wú)臨床癥狀出現(xiàn),每48小時(shí)稱量1次小鼠的體重變化。首次免疫后,分別于第28天,第56天進(jìn)行加強(qiáng)免疫。最后1次免疫后第7天處死兩組小鼠,并取下小鼠腓腸肌,制作冰凍切片。切片用四甲基羅丹明結(jié)合的銀環(huán)蛇毒素定位乙酰膽堿受體(acetylcholine receptor,AChR)(紅色),用FITC結(jié)合的羊抗小鼠IgG抗體定位抗LRP4抗體(綠色),在激光共聚焦顯微鏡下觀察。同時(shí)用透射電鏡觀察NMJ的超微結(jié)構(gòu)變化。結(jié)果:實(shí)驗(yàn)組與對(duì)照組小鼠未出現(xiàn)抓握和嘶叫無(wú)力的明顯臨床癥狀。實(shí)驗(yàn)組1只小鼠出現(xiàn)眼睛無(wú)法睜開(kāi)的臨床表現(xiàn)。激光共聚焦顯微鏡觀察后發(fā)現(xiàn),實(shí)驗(yàn)組腓腸肌的細(xì)胞膜表面存在綠色熒光,同一位置存在紅色熒光,表明抗LRP4抗體已經(jīng)與抗原LRP4結(jié)合,而且結(jié)合部位為NMJ的AChR處。透射電鏡觀察實(shí)驗(yàn)組小鼠肌細(xì)胞發(fā)現(xiàn),突觸結(jié)構(gòu)形態(tài)不規(guī)則,分支簡(jiǎn)化且分散,符合MG的病理變化特點(diǎn)。而對(duì)照組突觸結(jié)構(gòu)分支復(fù)雜且連續(xù),神經(jīng)末梢分支廣泛。結(jié)論:抗LRP4抗體已經(jīng)結(jié)合在NMJ突觸后膜的AChR部位的抗原LRP4上,導(dǎo)致突觸后膜出現(xiàn)超微結(jié)構(gòu)病理改變,并引起小鼠眼肌無(wú)力的臨床表現(xiàn),初步證明LRP4可以誘導(dǎo)小鼠MG,抗LRP4抗體是MG的致病性抗體。
[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.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R746.1

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相關(guān)期刊論文 前1條

1 劉睿;王桂平;杜嬰;周瓊;苗建亭;李柱一;;SD大鼠重癥肌無(wú)力被動(dòng)轉(zhuǎn)移模型的建立[J];中國(guó)神經(jīng)免疫學(xué)和神經(jīng)病學(xué)雜志;2012年01期

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本文編號(hào)：1643865