非神经元型胆碱能系统在重症肌无力患者外周血单个核细胞中的表达

发布时间：2018-06-15 04:26

本文选题：重症肌无力 + 外周血单个核细胞　；参考：《郑州大学》2017年硕士论文

【摘要】：目的重症肌无力(myasthenia gravis,MG)是比较常见的神经肌肉疾病,是细胞免疫依赖性、补体参与和自身反应抗体介导引起神经肌接头(neuromuscular junction,NMJ)处传递障碍的自身免疫性疾病,自身抗体作用的靶点为NMJ处突触后膜上乙酰胆碱受体(acetylcholine receptor,AChR)。重症肌无力患者血清中存在多种自身抗体,其中约85%为乙酰胆碱受体抗体(acetylcholine receptor antibody,AChRab),其余的自身抗体包括:肌肉特异性激酶抗体(muscle specific kinase antibody,MuSKab),低密度脂蛋白受体抗体(low density lipoprotein antibody,LRP-4ab),以及其他自身抗体。由于AChRab与AChR结合,使后者发生降解,从而引起骨骼肌反复活动后易疲劳症状。在前期研究中,免疫组化发现MG患者伴胸腺增生组与正常对照组相比,烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor,nAChR)表达减少,且在针对MG胸腺非神经元型胆碱能系统(non-neuronal cholinergic system,NNCS)相关组分分析时发现,表达存在明显差异(α1,α9,M3,α2,β2)。胸腺是自身反应性T细胞发育的器官,正常人外周血中成熟的单个核细胞存在NNCS,差异表达的NNCS组分会影响机体免疫和抗体的产生。本研究旨在观察其是否与胸腺表达NNCS存在联系,分析MG患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC)NNCS的表达,探讨其对自身抗体产生的影响。方法选取20例无吸烟史的健康志愿者(男10例,女10例)和30例来自郑州大学第二附属医院和河南省医药科学研究院MG患者(男13例,女17例),年龄25~57(39.4±10.4)岁,抽取肘正中静脉血6ml置于肝素抗凝管中,MG患者仅服用溴吡斯的明片或者未曾服用药物,不伴其他自身免疫性疾病。采集标本时取得患者的同意,所有患者均签署知情同意书。1.反转录PCR分析mAChR亚型(M1R,M2R,M3R,M4R,M5R),nAChR亚型(α2,α3,α4,α5,α6,α7,α9,α10,β2,β3,β4),胆碱乙酰转移酶(choline acetyltransferase,ChAT)和乙酰胆碱酯酶(acetylcholinesterase,AChE)的表达:采用Trizol法提取外周血单个核细胞总RNA,测定RNA的纯度和浓度,琼脂糖电泳观察RNA的完整性。根据反转录试剂盒将RNA反转录成cDNA,在一定条件下PCR仪中扩增,取5ul扩增产物行琼脂糖电泳跑胶,用凝胶图像分析仪拍照并分析电泳条带;2.Real-time PCR分析mAChR(M1R,M3R,M5R),nAChR(α5,α7)和AChE的表达:取上一步骤中cDNA,在定量PCR仪中一定条件下扩增,运用2-△△Ct法在MG组和正常对照组之间进行相对定量比较;3.统计学方法:应用SPSS 21.0统计软件分析,应用单因素方差分析和t检验比较MG组和对照组NNCS表达的差异,以p0.05为差异有统计学意义。结果1.RT-PCR分析MG组和对照组PBMC中M1R,M2R,M3R,M4R,M5R,α2,α3,α4,α5,α6,α7,α9,α10,β2,β3,β4,ChAT,AChE的表达:MG患者和对照组PBMC中均表达M1R,M3R,M5R,α5,α7,β2和AChE(均见明显条带),但是nAChR(α3,α9,β2)只在对照组中检测到,其余各亚型和ChAT在两组均未见表达(未见明显条带)。2.Real-time PCR分析MG组和对照组PBMC中mAChR(M1R,M3R,M5R),nAChR(α5,α7)和AChE的表达:M1 mAChR表达量为对照组的0.8倍(p=0.418),M3 mAChR为对照组的0.4倍(p=0.004),M5 m AChR为对照组的1.23倍(p=0.488),α5 nAChR为对照组的0.63倍(p=0.177),α7 nAChR为对照组的0.81倍(p=0.668),AChE为对照组的3.24倍(p=0.002),M3 mAChR表达显著低于对照组,AChE表达表达显著高于对照组。结论MG患者PBMC中nAChR(α3,α9,β2),M3 m AChR以及AChE表达存在差异,胸腺作为中枢免疫器官,主要是T细胞发育的场所,发育成熟的淋巴细胞通过外周血迁移进入淋巴器官发挥免疫效应,这种差异性表达可能是在胸腺T细胞发育过程中NNCS发生改变,进一步验证了胸腺在MG发病过程起到关键作用。
[Abstract]:Objective myasthenia gravis (myasthenia gravis, MG) is a relatively common neuromuscular disease. It is an autoimmune disease with cellular immune dependence, complement involvement and autoantibody mediated antibody mediated transfer of neuromuscular junction (neuromuscular junction, NMJ). The target of self antibody action is the acetylcholine receptor on the postsynaptic membrane of NMJ. The body (acetylcholine receptor, AChR). There are a variety of autoantibodies in the serum of myasthenia gravis, about 85% of which are acetylcholine receptor antibody (acetylcholine receptor antibody, AChRab), and the rest of the autoantibodies include: muscle specific kinase antibody (muscle specific kinase antibody, MuSKab), and low density lipoprotein receptor antibody. Density lipoprotein antibody, LRP-4ab), and other autoantibodies. The combination of AChRab and AChR causes the latter to degrade and cause the fatigue symptoms of the skeletal muscle after repeated activities. In the earlier study, the immunohistochemical discovery of the MG patients with the thymic hyperplasia group was compared with the normal control group, and the nicotinic acetylcholine receptor (nicotinic acetylchol). Ine receptor, nAChR) expression decreased, and in the analysis of the MG thymus non neuronal cholinergic system (non-neuronal cholinergic system, NNCS) related components analysis, the expression was significantly different (alpha 1, alpha 9, M3, alpha 2, beta 2). Thymus is a self reactive T cell hair organ, normal human peripheral blood mature mononuclear cells exist NNCS, poor The NNCS group of different expression affects the body immunity and the production of antibodies. The purpose of this study was to observe whether it was associated with the thymus expression of NNCS, and to analyze the expression of NNCS in peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) in MG patients, and to explore the effect on the production of autoantibodies. Methods 20 cases of no smoking history were selected. The participants (10 men, 10 women) and 30 MG patients (13 men, 17 women) from the Second Affiliated Hospital of Zhengzhou University and the Henan Academy of Medical Sciences (13 men, 17 women), 25~57 (39.4 + 10.4) years old, were extracted from Zhou Zhengzhong venous blood 6ml in heparin anticoagulant tube, MG patients only took Pyridostigmine Bromide Tablets or did not take drugs, without other autoimmune diseases. Disease. When the specimen was collected, all patients signed the consent of the patient, all patients signed the informed consent book.1. reverse transcriptional PCR analysis mAChR subtype (M1R, M2R, M3R, M4R, M5R), nAChR subtype (alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, alpha 9, alpha 10, beta 2, beta 3, beta 4), choline acetyl transferase (choline acetyltransferase, ChAT) and acetylcholinesterase expression Trizol method was used to extract the total RNA of peripheral blood mononuclear cells, to determine the purity and concentration of RNA, and to observe the integrity of RNA by agarose electrophoresis. According to the reverse transcriptional kit, RNA was transcribed into cDNA, and the PCR instrument was amplified by the PCR instrument under certain conditions. The gel was used for the agarose gel electrophoresis, and the gel image analyzer was used to photograph and analyze the electrophoresis strip; 2.Rea L-time PCR analysis mAChR (M1R, M3R, M5R), nAChR (alpha 5, alpha 7) and AChE expression: take a step in the cDNA, amplify under certain conditions in a quantitative PCR instrument, use 2- Delta Ct method to compare the relative quantitative comparison between the group and the normal control group; 3. statistical method: applying the 21 statistical software analysis, the application of single factor analysis of variance and the ratio test ratio The differences in the expression of NNCS in the MG group and the control group were statistically significant. Results 1.RT-PCR analyzed the M1R, M2R, M3R, M4R, M5R, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, alpha 9, alpha 7, alpha 9, alpha 7, alpha 9, alpha 10, beta 2, beta 3, beta 4, ChAT, and ChAT. ChR (alpha 3, alpha 9, beta 2) was detected only in the control group, and the other subtypes and ChAT were not expressed in the two groups (no obvious strip), and the mAChR (M1R, M3R, M5R) in the MG group and the control group PBMC, nAChR (alpha 5, alpha 7) and AChE, were 0.8 times that of the control group and 0.4 times of the control group. ChR was 1.23 times (p=0.488), alpha 5 nAChR was 0.63 times as the control group (p=0.177), alpha 7 nAChR was 0.81 times as the control group (p=0.668), AChE was 3.24 times of the control group (p=0.002), M3 mAChR expression was significantly lower than the control group, AChE expression was significantly higher than that of the control group. The thymus is the central immune organ, which is mainly the place of T cell development. The mature lymphocytes migrate through the peripheral blood into the lymphoid organs to play the immune effect. This differential expression may be changed in the development of thymus T cells, and further proves that the thymus plays a key role in the pathogenesis of MG.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R746.1

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