本文選題：重癥肌無(wú)力 + 外周血單個(gè)核細(xì)胞　； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：目的重癥肌無(wú)力(myasthenia gravis,MG)是比較常見(jiàn)的神經(jīng)肌肉疾病,是細(xì)胞免疫依賴性、補(bǔ)體參與和自身反應(yīng)抗體介導(dǎo)引起神經(jīng)肌接頭(neuromuscular junction,NMJ)處傳遞障礙的自身免疫性疾病,自身抗體作用的靶點(diǎn)為NMJ處突觸后膜上乙酰膽堿受體(acetylcholine receptor,AChR)。重癥肌無(wú)力患者血清中存在多種自身抗體,其中約85%為乙酰膽堿受體抗體(acetylcholine receptor antibody,AChRab),其余的自身抗體包括:肌肉特異性激酶抗體(muscle specific kinase antibody,MuSKab),低密度脂蛋白受體抗體(low density lipoprotein antibody,LRP-4ab),以及其他自身抗體。由于AChRab與AChR結(jié)合,使后者發(fā)生降解,從而引起骨骼肌反復(fù)活動(dòng)后易疲勞癥狀。在前期研究中,免疫組化發(fā)現(xiàn)MG患者伴胸腺增生組與正常對(duì)照組相比,煙堿型乙酰膽堿受體(nicotinic acetylcholine receptor,nAChR)表達(dá)減少,且在針對(duì)MG胸腺非神經(jīng)元型膽堿能系統(tǒng)(non-neuronal cholinergic system,NNCS)相關(guān)組分分析時(shí)發(fā)現(xiàn),表達(dá)存在明顯差異(α1,α9,M3,α2,β2)。胸腺是自身反應(yīng)性T細(xì)胞發(fā)育的器官,正常人外周血中成熟的單個(gè)核細(xì)胞存在NNCS,差異表達(dá)的NNCS組分會(huì)影響機(jī)體免疫和抗體的產(chǎn)生。本研究旨在觀察其是否與胸腺表達(dá)NNCS存在聯(lián)系,分析MG患者外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell,PBMC)NNCS的表達(dá),探討其對(duì)自身抗體產(chǎn)生的影響。方法選取20例無(wú)吸煙史的健康志愿者(男10例,女10例)和30例來(lái)自鄭州大學(xué)第二附屬醫(yī)院和河南省醫(yī)藥科學(xué)研究院MG患者(男13例,女17例),年齡25~57(39.4±10.4)歲,抽取肘正中靜脈血6ml置于肝素抗凝管中,MG患者僅服用溴吡斯的明片或者未曾服用藥物,不伴其他自身免疫性疾病。采集標(biāo)本時(shí)取得患者的同意,所有患者均簽署知情同意書(shū)。1.反轉(zhuǎn)錄PCR分析mAChR亞型(M1R,M2R,M3R,M4R,M5R),nAChR亞型(α2,α3,α4,α5,α6,α7,α9,α10,β2,β3,β4),膽堿乙酰轉(zhuǎn)移酶(choline acetyltransferase,ChAT)和乙酰膽堿酯酶(acetylcholinesterase,AChE)的表達(dá):采用Trizol法提取外周血單個(gè)核細(xì)胞總RNA,測(cè)定RNA的純度和濃度,瓊脂糖電泳觀察RNA的完整性。根據(jù)反轉(zhuǎn)錄試劑盒將RNA反轉(zhuǎn)錄成cDNA,在一定條件下PCR儀中擴(kuò)增,取5ul擴(kuò)增產(chǎn)物行瓊脂糖電泳跑膠,用凝膠圖像分析儀拍照并分析電泳條帶;2.Real-time PCR分析mAChR(M1R,M3R,M5R),nAChR(α5,α7)和AChE的表達(dá):取上一步驟中cDNA,在定量PCR儀中一定條件下擴(kuò)增,運(yùn)用2-△△Ct法在MG組和正常對(duì)照組之間進(jìn)行相對(duì)定量比較;3.統(tǒng)計(jì)學(xué)方法:應(yīng)用SPSS 21.0統(tǒng)計(jì)軟件分析,應(yīng)用單因素方差分析和t檢驗(yàn)比較MG組和對(duì)照組NNCS表達(dá)的差異,以p0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果1.RT-PCR分析MG組和對(duì)照組PBMC中M1R,M2R,M3R,M4R,M5R,α2,α3,α4,α5,α6,α7,α9,α10,β2,β3,β4,ChAT,AChE的表達(dá):MG患者和對(duì)照組PBMC中均表達(dá)M1R,M3R,M5R,α5,α7,β2和AChE(均見(jiàn)明顯條帶),但是nAChR(α3,α9,β2)只在對(duì)照組中檢測(cè)到,其余各亞型和ChAT在兩組均未見(jiàn)表達(dá)(未見(jiàn)明顯條帶)。2.Real-time PCR分析MG組和對(duì)照組PBMC中mAChR(M1R,M3R,M5R),nAChR(α5,α7)和AChE的表達(dá):M1 mAChR表達(dá)量為對(duì)照組的0.8倍(p=0.418),M3 mAChR為對(duì)照組的0.4倍(p=0.004),M5 m AChR為對(duì)照組的1.23倍(p=0.488),α5 nAChR為對(duì)照組的0.63倍(p=0.177),α7 nAChR為對(duì)照組的0.81倍(p=0.668),AChE為對(duì)照組的3.24倍(p=0.002),M3 mAChR表達(dá)顯著低于對(duì)照組,AChE表達(dá)表達(dá)顯著高于對(duì)照組。結(jié)論MG患者PBMC中nAChR(α3,α9,β2),M3 m AChR以及AChE表達(dá)存在差異,胸腺作為中樞免疫器官,主要是T細(xì)胞發(fā)育的場(chǎng)所,發(fā)育成熟的淋巴細(xì)胞通過(guò)外周血遷移進(jìn)入淋巴器官發(fā)揮免疫效應(yīng),這種差異性表達(dá)可能是在胸腺T細(xì)胞發(fā)育過(guò)程中NNCS發(fā)生改變,進(jìn)一步驗(yàn)證了胸腺在MG發(fā)病過(guò)程起到關(guān)鍵作用。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R746.1

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