基因干预MST2的施万细胞稳转株建立及其在线粒体膜电位检测的应用  

Establishment of stable Schwann cell lines with genetic intervention of MST2 and its appliance in the detec⁃tion of mitochondrial membrane potential

作　　者：黄贝旭 刘昶 梁嫩 廖松洁[1] HUANG Beixu;LIU Chang;LIANG Nen;LIAO Songjie(Department of Neurology,The First Affiliated Hospital,Sun Yat-sen University,Guangdong Provincial Key Laboratory of Diagno-sis and Treatment of Major Neurological Diseases,National Key Clinical Department and Key Discipline of Neurol-ogy,Guangzhou,510080,China)

机构地区：[1]中山大学附属第一医院神经科,广东省重大疾病诊治研究重点实验室,国家临床重点专科和国家重点学科,广州510080

出　　处：《中国神经精神疾病杂志》2024年第2期102-109,共8页Chinese Journal of Nervous and Mental Diseases

基　　金：国家自然科学基金面上项目(编号:82071366);广州市临床特色技术项目(编号:2023P-TS21);广东省自然科学基金面上项目(编号:2023A1515010486);广东省神经系统疾病临床医学研究中心(编号:2020B1111170002);广东省神经疾病早期干预及功能修复研究国际科技合作基地(编号:2020A0505020004);广东省神经系统重大疾病诊治工程技术研究中心、广东省神经系统重大疾病诊治转化医学创新平台和广州市神经系统重大疾病临床医学研究与转化中心项目资助。

摘　　要：目的构建稳定敲低及过表达哺乳动物Ste20样激酶2(mammalian ste20-like kinase 2, MST2)的大鼠施万细胞株,并初步探讨MST2对线粒体膜电位的调控作用。方法 大鼠施万细胞株(RSC96)分为正常对照组(对照组)、氧糖剥夺(oxygen and glucose deprivation, OGD)模型组、MST2敲低对照组、MST2敲低组、MST2过表达对照组、MST2过表达组。逆转录PCR法扩增大鼠Mst2基因全长序列,构建过表达Mst2基因的慢病毒表达载体重组质粒并鉴定。使用第二代慢病毒包装系统获取Mst2基因敲低及过表达的慢病毒毒液,并分别感染RSC96细胞株,建立基因干预MST2的稳转株。实时荧光定量PCR结合western blot检测基因干预效率,光镜结合S100荧光染色观察稳转株形态学变化。使用OGD模型处理细胞,以线粒体膜电位检测试剂盒(JC-1)检测细胞线粒体膜电位水平(mitochondrial membrane potential, MMP)。结果 经测序分析,成功构建Mst2慢病毒表达载体重组质粒。施万细胞稳转株具有施万细胞的形态特征,并且表达施万细胞特异性标志物S100。在慢病毒感染的施万细胞,MST2表达在mRNA及蛋白水平上均较对照组有稳定且显著的差异,敲低效率超过90%,过表达约为对照组的40倍。稳转株在OGD模型中,对于MST2蛋白的基因干预仍然维持稳定。OGD可显著增加MST2蛋白表达,并降低施万细胞MMP,提示线粒体功能受损;而敲低MST2则显著改善MMP。结论 本研究成功构建了稳定敲低及过表达MST2的大鼠施万细胞株,并且在OGD模型中,敲低MST2可改善线粒体膜电位,提示其对线粒体具有保护作用。Objective To construct rat Schwann cell RSC96 cell lines that stably knock down or overexpress mammalian ste20-like kinase 2(MST2),and preliminarily explore its regulatory effect on mitochondrial membrane potential.Methods The rat Schwann cell lines(RSC96)were divided into normal control group,oxygen and glucose deprivation(OGD)model group,MST2 knockdown control group,MST2 knockdown group,MST2 overexpression control group,MST2 overexpression group.The full-length sequence of rat Mst2 gene was amplified by reverse transcription PCR,and then a recombinant plasmid of overexpressing Mst2 lentiviral expression vector was constructed and identified.The second-generation lentiviral packaging system was used to obtain Mst2 knockdown or overexpression lentiviral venom and then infected the RSC96 cell lines to establish stably transfected cell lines with genetic intervention of MST2.Real-time fluorescence quantitative PCR combined with western blot was used to detect the efficiency of gene intervention.Light microscopy combined with S100 fluorescent staining was used to examine the morphological changes of the cell lines.Cells were exposed to the OGD,and the mitochondrial membrane potential(MMP)level of the cells was detected using a mitochondrial membrane potential detection kit(JC-1).Results The Mst2 lentiviral expression vector recombinant plasmid was successfully constructed by using plasmid sequencing.The stably transfected Schwann cell lines had the morphological characteristics of Schwann cells and expressed the Schwann cell-specific marker S100.In lentivirus-infected Schwann cells,MST2 expression was stably and significantly different at both the mRNA and protein levels compared with the control group.The knockdown efficiency exceeded 90%and overexpression was approximately 40 times that of the control.In the OGD model,the genetic intervention of MST2 remained stable as well.OGD could significantly increase MST2 and reduce Schwann cell MMP,indicating the impairment of mitochondrial function while knocking down MST2

关 键 词：MST2 施万细胞 稳转株 氧糖剥夺 线粒体膜电位 

分 类 号：R741.02[医药卫生—神经病学与精神病学] R338[医药卫生—临床医学]