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簇集表达的乙酰胆碱受体在重症肌无力抗体检测中的应用

发布时间:2018-04-14 17:32

  本文选题:重症肌无力 + 簇集表达的乙酰胆碱受体抗体 ; 参考:《第二军医大学》2014年硕士论文


【摘要】:目的 一、ELISA法检测24例重症肌无力患者血清乙酰胆碱受体抗体水平,筛查血清乙酰胆碱受体抗体阳性及阴性患者,为后续试验做好准备; 二、将成人骨骼肌细胞烟碱型乙酰胆碱受体各亚基以及促使其聚集的缔合蛋白RAPSN在人胚肾细胞(HEK293T)表面共表达,从而建立基于细胞的重症肌无力乙酰胆碱受体抗体检测模型,通过该模型检验24例患者血清乙酰胆碱受体抗体,比较两种方法的检验结果。 方法 一、收集我科24例重症肌无力患者,通过酶联免疫吸附法(ELISA法)检测血清乙酰胆碱受体抗体的含量,筛查乙酰胆碱受体抗体阳性及阴性患者。 二、分别从含有AchR亚基α、β、δ、ε以及Rapsn五种质粒的菌液中,将目的基因的质粒提取。将AchR亚基α、β、δ、ε质粒,Rapsyn质粒按一定比例转染细胞,使其共表达在细胞表面。再通过免疫荧光法检测血清乙酰胆碱受体抗体,并与ELISA结果进行比较。 三、检测指标 (一)ELISA样品的吸光度; (二)琼脂糖电泳及基因测序验证目的基因片段; (三)western blot方法检测各细胞转染后蛋白表达情况; (四)荧光显微镜观察转染后荧光蛋白的表达与共定位情况; (五)对比两种方法检验结果。 结果 一、ELISA法:根据30例非重症肌无力志愿者ELISA结果,推测血清乙酰胆碱受体抗体吸光度可疑阳性范围:0.249-0.272,阳性范围:大于0.272。据此得出24例患者,ELISA阳性3例,可疑阳性4例,阴性17例。 二、琼脂糖电泳证实质粒RAPSN-EGFP片段与目的基因相符合。五种质粒基因测序结果均与目的质粒相符合。western blot显示目的条带的大小相符合。 三、荧光显微镜显示,转染了AchR各亚基、RAPSN质粒的细胞绿色荧光呈颗粒状并聚集于细胞膜。 四、细胞模型检验24例患者血清,阳性能够见到明显的免疫荧光共定位现象。 五、细胞模型免疫荧光法检测24例患者总阳性率70.83%,与ELISA法相比,结果有统计学意义(P0.05)。ELISA阴性患者17例中,免疫荧光法阳性率58.82%。 结论 一、ELISA检验是目前重症肌无力患者乙酰胆碱受体抗体检验的常用方法,,但其敏感性有限。 二、转染RAPSN质粒的细胞实现了乙酰胆碱受体的簇集表达。经过初步检测,认为该模型检验血清中乙酰胆碱受体抗体阳性率高于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 伪, 尾, 未, 蔚 and Rapsn, respectively.The AchR subunits 伪, 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.
【学位授予单位】:第二军医大学
【学位级别】:硕士
【学位授予年份】:2014
【分类号】:R746.1

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