α7烟碱型乙酰胆碱受体在重症肌无力胸腺细胞中的表达及其功能  

Expression and function ofα7 nicotinic acetylcholine receptor in thymocytes of myasthenia gravis patients

作　　者：黄玉炜 王猛 张永辉 崔新征 孙子瑞 张志文 史宸硕 张清勇 HUANG Yuwei;WANG Meng;ZHANG Yonghui;CUI Xinzheng;SUN Zirui;ZHANG Zhiwen;SHI Chenshuo;ZHANG Qingyong(Myasthenia Gravis Comprehensive Diagnosis and Treatment Center,Henan Provincial People’s Hospital,Zhengzhou,450003,P.R.China;Comprehensive Laboratory,Henan Provincial People’s Hospital,Zhengzhou,450003,P.R.China;Department of Adult Cardiac Surgery,Fuwai Huazhong Cardiovascular Hospital,Zhengzhou,450003,P.R.China)

机构地区：[1]河南省人民医院重症肌无力综合诊疗中心,郑州450003 [2]河南省人民医院综合实验室,郑州450003 [3]阜外华中心血管病医院成人心外科,郑州450003

出　　处：《中国胸心血管外科临床杂志》2023年第6期897-902,共6页Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

基　　金：河南省科技攻关项目(212102310153)。

摘　　要：目的探讨重症肌无力(myasthenia gravis,MG)患者胸腺细胞中α7烟碱型乙酰胆碱受体(α7nicotinic acetylcholine receptor,α7 nAChR)的表达及其对胸腺细胞分泌细胞因子和T细胞增殖的影响。方法选取2021年6月—2022年6月河南省人民医院重症肌无力综合诊疗中心收治的行胸腔镜下胸腺扩大切除术MG患者为MG组,另选取阜外华中心血管病医院成人心外科2021年6月—2022年9月行心脏疾病手术过程中为暴露术野行部分胸腺切除术的患者为对照组。制备MG组及对照组胸腺单细胞悬液,采用实时荧光聚合酶链式反应、蛋白免疫印迹法检测患者胸腺细胞中α7 nAChR的表达情况。采用CD3/CD28单抗偶联磁珠诱导T细胞活化,酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测胸腺细胞中细胞因子干扰素-γ(interferongamma,IFN-γ)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-4(interleukin-4,IL-4)、IL-6、IL-10、IL-17、IL-21表达水平。按处理方式不同,将活化的MG组胸腺T细胞分为空白对照组、α7 nAChR拮抗剂组、α7 nAChR激动剂组。培养72 h后采用ELISA检测培养上清中IFN-γ、TNF-α、IL-4、IL-6、IL-10、IL-17、IL-21表达水平。之后加入CD4-PE、CD8-APC抗体,采用流式细胞术检测T细胞亚群增殖情况。结果共纳入10例MG患者,其中男3例、女7例,平均年龄(19.25±6.28)岁。纳入对照组患者15例,其中男6例、女9例,平均年龄(26.18±6.77)岁。与对照组相比,MG组胸腺细胞中α7 nAChR mRNA及蛋白表达水平降低(P<0.001),细胞因子IFN-γ、TNF-α、IL-4、IL-6、IL-21表达水平升高(P<0.05),IL-10、IL-17表达差异无统计学意义(P>0.05)。细胞培养实验结果显示,与空白对照组相比,α7 nAChR拮抗剂组胸腺T细胞分泌IFN-γ、TNF-α、IL-6、IL-21水平升高(P<0.05),而α7 nAChR激动剂组降低(P<0.05),三组IL-4、IL-10、IL-17分泌水平差异无统计学意义(P>0.05)。α7 nAChR激动剂组CD4+T、CD8+T细胞计Objective To investigate the expression ofα7 nicotinic acetylcholine receptor(α7 nAChR)in thymocytes of patients with myasthenia gravis(MG)and its effect on cytokine secretion and T cell proliferation.Methods Patients with MG who underwent expanded thoracoscopic thymectomy in the Comprehensive Diagnosis and Treatment Center of the Henan Provincial People’s Hospital from June 2021 to June 2022 were selected and allocated to a MG group.Patients who underwent partial thymectomy to expose the surgical field during the cardiac disease surgery from June 2021 to September 2022 in the Department of Adult Cardiac Surgery of Fuwai Huazhong Cardiovascular Hospital were selected as the control group.Thymic single cell suspensions were prepared from MG and control groups,and the expression ofα7 nAChR in thymocytes of the two groups was detected by real-time polymerase chain reaction and Western blotting.Then CD3/CD28 monoclonal antibody coupled with magnetic beads was used to induce T cell activation,and the levels of cytokines interferon-gamma(IFN-γ),tumor necrosis factor-α(TNF-α),interleukin-4(IL-4),IL-6,IL-10,IL-17,and IL-21 in thymocytes of the two groups were detected by enzyme-linked immunosorbent assay(ELISA).The activated T cells of the MG group were divided into a blank control group,anα7 nAChR antagonist group,and anα7 nAChR agonist group according to different treatment methods.After 72 hours of culture,IFN-γ,TNF-α,IL-4,IL-6,IL-10,IL-17,and IL-21 expression levels in the culture supernatant were measured by ELISA.Afterwards,CD4-PE and CD8-APC antibodies were added,and the proliferation of T cell subsets was detected by flow cytometry.Results A total of 10 MG patients were collected,including 3 males and 7 females with an average age of 19.25±6.28 years;and 15 control patients were collected,including 6 males and 9 females with an average age of 26.18±6.77 years.Compared with the control group,the mRNA and protein levels ofα7 nAChR in the thymocytes of MG group were decreased,and the expression levels

关 键 词：Α7烟碱型乙酰胆碱受体 重症肌无力 细胞因子 基础研究 

分 类 号：R746.1[医药卫生—神经病学与精神病学]