本文選題：重癥肌無力 + 簇集表達的乙酰膽堿受體抗體�。� 參考：《第二軍醫(yī)大學》2014年碩士論文

【摘要】：目的 一、ELISA法檢測24例重癥肌無力患者血清乙酰膽堿受體抗體水平，篩查血清乙酰膽堿受體抗體陽性及陰性患者，為后續(xù)試驗做好準備； 二、將成人骨骼肌細胞煙堿型乙酰膽堿受體各亞基以及促使其聚集的締合蛋白RAPSN在人胚腎細胞（HEK293T）表面共表達，從而建立基于細胞的重癥肌無力乙酰膽堿受體抗體檢測模型，通過該模型檢驗24例患者血清乙酰膽堿受體抗體，比較兩種方法的檢驗結(jié)果。 方法 一、收集我科24例重癥肌無力患者，通過酶聯(lián)免疫吸附法（ELISA法）檢測血清乙酰膽堿受體抗體的含量，篩查乙酰膽堿受體抗體陽性及陰性患者。 二、分別從含有AchR亞基α、β、δ、ε以及Rapsn五種質(zhì)粒的菌液中，將目的基因的質(zhì)粒提取。將AchR亞基α、β、δ、ε質(zhì)粒，Rapsyn質(zhì)粒按一定比例轉(zhuǎn)染細胞，使其共表達在細胞表面。再通過免疫熒光法檢測血清乙酰膽堿受體抗體，并與ELISA結(jié)果進行比較。 三、檢測指標 （一）ELISA樣品的吸光度； （二）瓊脂糖電泳及基因測序驗證目的基因片段； （三）western blot方法檢測各細胞轉(zhuǎn)染后蛋白表達情況； （四）熒光顯微鏡觀察轉(zhuǎn)染后熒光蛋白的表達與共定位情況； （五）對比兩種方法檢驗結(jié)果。 結(jié)果 一、ELISA法：根據(jù)30例非重癥肌無力志愿者ELISA結(jié)果，推測血清乙酰膽堿受體抗體吸光度可疑陽性范圍：0.249-0.272，陽性范圍：大于0.272。據(jù)此得出24例患者，ELISA陽性3例，可疑陽性4例，陰性17例。 二、瓊脂糖電泳證實質(zhì)粒RAPSN-EGFP片段與目的基因相符合。五種質(zhì)�；驕y序結(jié)果均與目的質(zhì)粒相符合。western blot顯示目的條帶的大小相符合。 三、熒光顯微鏡顯示，轉(zhuǎn)染了AchR各亞基、RAPSN質(zhì)粒的細胞綠色熒光呈顆粒狀并聚集于細胞膜。 四、細胞模型檢驗24例患者血清，陽性能夠見到明顯的免疫熒光共定位現(xiàn)象。 五、細胞模型免疫熒光法檢測24例患者總陽性率70.83%，與ELISA法相比，結(jié)果有統(tǒng)計學意義（P0.05）。ELISA陰性患者17例中，免疫熒光法陽性率58.82%。 結(jié)論 一、ELISA檢驗是目前重癥肌無力患者乙酰膽堿受體抗體檢驗的常用方法，，但其敏感性有限。 二、轉(zhuǎn)染RAPSN質(zhì)粒的細胞實現(xiàn)了乙酰膽堿受體的簇集表達。經(jīng)過初步檢測，認為該模型檢驗血清中乙酰膽堿受體抗體陽性率高于ELISA法。
[Abstract]:PurposeThe level of serum acetylcholine receptor antibody was detected by Elisa in 24 patients with myasthenia gravis.Secondly, the nicotinic acetylcholine receptor subunits of adult skeletal muscle cells and the association protein RAPSN, which promotes its aggregation, were co-expressed on the surface of human embryonic kidney cells (HEK293T), so as to establish a cell-based model for the detection of acetylcholine receptor antibodies against myasthenia gravis.The serum acetylcholine receptor antibodies in 24 patients were detected by this model and the results of the two methods were compared.MethodOne, 24 patients with myasthenia gravis in our department were collected. The serum levels of serum acetylcholine receptor antibodies were detected by Elisa, and the positive and negative patients were screened.Secondly, the plasmid of the target gene was extracted from the liquid containing five plasmids of AchR subunit 偽, 尾, 未, 蔚 and Rapsn, respectively.The AchR subunits 偽, 尾, 未, 蔚 plasmids were transfected with Rapsyn plasmid in a certain proportion and coexpressed on the cell surface.Then the serum acetylcholine receptor antibody was detected by immunofluorescence method and compared with the results of ELISA.III. Testing indicators(a) absorbance of Elisa samples;(2) agarose electrophoresis and gene sequencing;(3) Western blot method was used to detect the expression of protein after transfection.(4) the expression and co-localization of fluorescent protein after transfection were observed by fluorescence microscope.(5) compare the results of the two methods.ResultElisa: according to the ELISA results of 30 non-myasthenia gravis volunteers, we speculated that the serum acetylcholine receptor antibody absorbency was suspected to be in the range of 0.249-0.272, and the positive range was greater than 0.272.According to the results, 3 cases were positive for Elisa, 4 cases were suspicious positive, and 17 cases were negative.Secondly, agarose electrophoresis confirmed that the plasmid RAPSN-EGFP fragment was consistent with the target gene.The sequencing results of the five plasmids were consistent with the size of the target bands displayed by western. Blot.3. Fluorescence microscope showed that the green fluorescence of the cells transfected with AchR subunit RAPSN plasmids was granular and aggregated on the cell membrane.4. The positive immunofluorescence co-localization was observed in 24 cases of serum detected by cell model.Fifth, the total positive rate of immunofluorescence assay was 70.83% in 24 patients. Compared with ELISA method, the positive rate of immunofluorescence assay was 58.82% in 17 patients with negative P0.05. Elisa.ConclusionElisa is a common method for the detection of acetylcholine receptor antibodies in patients with myasthenia gravis, but its sensitivity is limited.Secondly, the cells transfected with RAPSN plasmid expressed acetylcholine receptor in clusters.The results showed that the positive rate of serum acetylcholine receptor antibody was higher than that of ELISA method.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R746.1

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