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0.969 6(95%CI:0.943 5～0.995 7)、0.944 0(95%CI:0.904 7～0.983 3)、0.855 6(95%CI:0.776 7～0.934 5)及 0.908 8(95%CI:0.852 6～0.965 0)(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.
Abstract
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 LC3Ⅱ mRNA 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 LC3Ⅱ were 0.969 6(95%CI:0.943 5-0.995 7),0.944 0(95%CI:0.904 7-0.983 3),0.855 6(95%CI:0.776 7-0.934 5),and 0.908 8(95%CI:0.852 6-0.965 0)(P<0.01),respectively,and the sensitivities were 92%,92%,78%,and 84%,and the specificities were 88%,82%,92%,and 82%,respectively;whereas the AUC for Cyt-C,Bax,Bcl-2,caspase 3,and caspase 9 was 1.000(P<0.01),and the sensitivity and specificity reached 100%.Conclusion:Mitochondrial autophagy disorder and increased apoptosis are found in the PBMCs in the patients with MG,which has a higher guiding value for clinical diagnosis of MG.
基金项目
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)
国家科技期刊平台
NSTL
维普
万方数据