重症肌无力患者外周血单个核细胞中线粒体自噬和凋亡相关基因表达及其诊断价值  

Expression of mitophagy and apoptosis related genes in peripheral blood mononuclear cells of patients with myasthenia gravis and its clinical diagnosis value

作　　者：蔡东红 李青 柯铃铃 钟慧雅 江其龙[2] 张涵 宋雅芳[1] CAI Donghong;LI Qing;KE Lingling;ZHONG Huiya;JIANG Qilong;ZHANG Han;SONG Yafang(Science and Technology Innovation Center,Institute of Spleen and Stomach Diseases,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;Department of Spleen and Stomach Diseases,First Affiliated Hospital,Guangzhou University of Chinese Medicine,Guangzhou 510405,China;School of Traditional Chinese Medicine,Guangdong Pharmaceutical University,Guangzhou 510405,China)

机构地区：[1]广州中医药大学科技创新中心广州中医药大学脾胃研究所,广东广州510405 [2]广州中医药大学第一附属医院脾胃病科,广东广州510405 [3]广东药科大学中药学院,广东广州510405

出　　处：《吉林大学学报（医学版）》2024年第2期481-488,共8页Journal of Jilin University：Medicine Edition

基　　金：国家自然科学基金面上项目(82374391);广东省科技厅自然科学基金面上项目(2023A1515011127);广东省教育厅生物医药与健康重点领域专项(2021ZDZX2032)。

摘　　要：目的:探讨重症肌无力(MG)患者外周血单个核细胞(PBMCs)中线粒体自噬和凋亡相关基因的表达水平,阐明其对MG诊断的指导价值。方法:收集并提取50名健康对照者(对照组)和50例MG患者(MG组)的PBMCs,采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测线粒体自噬因子磷脂酶和张力蛋白同源物(PTEN)诱导激酶1(PINK1)、E3泛素连接酶PARK2(Parkin)、泛素结合蛋白62(p62)、微管相关蛋白1轻链3(LC3Ⅱ)及凋亡因子细胞色素C(Cyt-C)、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、含半胱氨酸的天冬氨酸蛋白酶3(caspase 3)和含半胱氨酸的天冬氨酸蛋白酶9(caspase 9)的mRNA以及蛋白表达水平,根据mRNA检测结果设定多个不同临界值,在每个临界值处计算出相应的灵敏度和特异度,并绘制受试者工作特征(ROC)曲线。结果:与对照组比较,MG组患者PBMCs中的自噬因子PINK1、Parkin和LC3Ⅱ的mRNA及蛋白表达水平明显降低(P<0.01),p62 mRNA和蛋白表达水平升高(P<0.05或P<0.01);凋亡因子Cyt-C、Bax、caspase 3和caspase 9 mRNA及蛋白表达水平明显升高(P<0.05或P<0.01),Cyt-C mRNA和蛋白表达水平升高(P<0.05或P<0.01),而Bcl-2 mRNA和蛋白表达水平明显降低(P<0.01)。ROC曲线检测,PINK1、Parkin、p62和LC3Ⅱ的ROC曲线下面积(AUC)分别为0.9696(95%CI:0.9435~0.9957)、0.9440(95%CI:0.9047~0.9833)、0.8556(95%CI:0.7767~0.9345)及0.9088(95%CI:0.8526~0.9650)(P<0.01),灵敏度分别为92%、92%、78%和84%,特异度分别为88%、82%、92%和82%;而Cyt-C、Bax、Bcl-2、caspase 3和caspase 9的AUC均为1.000(P<0.01),灵敏度和特异度均达到100%。结论:MG患者PBMCs中存在线粒体自噬障碍和凋亡增加现象,其对临床诊断MG具有较高的指导价值。Objective:To discuss the expression levels of mitochondrial autophagy and apoptosis-related genes in peripheral blood mononuclear cells(PBMCs)in the patients with myasthenia gravis(MG),and to clarify its diagnostic value for MG.Methods:The PBMCs from 50 healthy controls(control group)and 50 MG patients(MG group)were collected and extracted.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of mitochondrial autophagy factors phosphatase and tensin homolog(PTEN)-induced kinase 1(PINK1),E3 ubiquitin-protein ligase PARK2(Parkin),ubiquitin-binding protein p62(p62),microtubule-associated proteins light chain 3 typeⅡ(LC3Ⅱ),and apoptotic factors cytochrome C(Cyt-C),B cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cysteine-containing aspartic acid protease 3(caspase 3),and cysteine-containing aspartic acid protease 9(caspase 9)mRNA and protein.Different cut-off values were set based on the mRNA detection results,and the corresponding sensitivity and specificity at each cut-off value were calculated.The receiver operating characteristic(ROC)curve was drawn.Results:Compared with control group,the expression levels of autophagy factors PINK1,Parkin,and LC3ⅡmRNA and protein in the PBMCs in MG group were significantly decreased(P<0.01),while the expression levels of p62 mRNA and protein were increased(P<0.05 or P<0.01).The expression levels of apoptotic factors Cyt-C,Bax,caspase 3,and caspase 9 mRNA and protein were significantly increased(P<0.05 or P<0.01),and while the expression levels of Cyt-C mRNA and protein were increased(P<0.05 or P<0.01),and the expression levels of Bcl-2 mRNA and protein were significantly decreased(P<0.01).The ROC curve analysis results showed that the area under the curve(AUC)for PINK1,Parkin,p62,and LC3Ⅱwere 0.9696(95%CI:0.9435-0.9957),0.9440(95%CI:0.9047-0.9833),0.8556(95%CI:0.7767-0.9345),and 0.9088(95%CI:0.8526-0.9650)(P<0.01),respectively,and the sensitivities were 92%,92%,78%,and 84%,and the specificities were

关 键 词：重症肌无力 线粒体自噬 细胞凋亡 外周血单个核细胞 诊断价值 

分 类 号：R746.1[医药卫生—神经病学与精神病学]