心肌致密化不全患者特异性诱导多潜能干细胞及心肌样细胞的构建  

Generation of induced pluripotent stem cells and cardiac myocyte-like cells from a patient with noncompaction of ventricular myocardium

作　　者：刘奥怡 张蕾[1] 龚梦嘉[1] 田杰[1] 吕铁伟[1] LIU Aoyi;ZHANG Lei;GONG Mengjia;TIAN Jie;LYU Tiewei(Department of Cardiology,Key Laboratory of Child Development and Disorders of Ministry of Education,National Clinical Research Center for Child Health and Disorders,International Science and Technology Cooperation Base of Child Development and Critical Disorders,Chongqing Key Laboratory of Pediatrics,Children's Hospital Affiliated to Chongqing Medical University,Chongqing,400014,China)

机构地区：[1]重庆医科大学附属儿童医院心血管内科儿童发育疾病研究教育部重点试验室国家儿童健康与疾病临床医学研究中心儿童发育重大疾病国家国际科技合作基地儿科学重庆市重点试验室

出　　处：《第三军医大学学报》2019年第24期2401-2408,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81570218)~~

摘　　要：目的构建心肌致密化不全(noncompaction of ventricular myocardium,NVM)患者来源的诱导多潜能干细胞(induced pluripotent stem cells,iPS)及心肌样细胞(cardiomyocytes,CMs)。方法用仙台病毒感染1名NVM患者(女性,16岁,2016年我科住院患者)和1名同期24岁健康男性志愿者的尿液细胞获得iPS细胞,进而将iPS细胞诱导分化为iPS-CMs。对iPS和iPS-CMs进行免疫荧光染色检测干细胞和心肌细胞标志物、碱性磷酸酶(alkaline phosphatase,AP)活性检测、核型分析、拟胚体(embryoidbodies,EBs)形成和膜片钳试验检测,同时采用全外显子测序(whole exon sequencing,WES)对NVM患者血液、iPS及iPS-CMs进行遗传学背景的比较,并筛选出可能和NVM发生相关的突变基因。结果成功获得NVM患者及正常人的尿液细胞和iPS,并分化为iPS-CMs。免疫荧光显示iPS表达干细胞标志Oct4、Sox2、Ssea4和TRA-1-81,iPS细胞具有高AP活性、具有正常核型、克隆能够在体外悬浮培养分化成3个胚层的细胞;iPS-CMs表达心肌细胞特异性标志物α-actinin和TNNT2,膜片钳结果显示iPS-CMs形成心肌细胞特异性钠电流及动作电位。WES结果显示患者血液、iPS及iPS-CMs三者具有相似的遗传谱,并发现可能与NVM发生相关的突变。结论成功构建出NVM患者的iPS-CMs,为采用iPS技术开展NVM的研究提供有力的细胞模型。Objective To obtain induced pluripotent stem cells(iPS)and cardiac myocyte-like cells(CMs)from a patient with noncompaction of ventricular myocardium(NVM).Methods Urine cells from a 16-year-old female patient with NVM and a 24-year-old healthy male volunteer were infected with Sendai virus to obtain iPS cells,which were further induced to differentiate into iPS-CMs.Both the iPS cells and iPS-CMs were identified by immunofluorescence staining,alkaline phosphatase activity,karyotype analysis,embryoid body(EBs)formation and cell patch-clamp experiment.Whole-exome sequencing of the blood sample,iPS cells and iPS-CMs from the NVM patient was performed for genetic comparison to identify gene mutations potentially related with NVM.Results We successfully obtained urine cells,iPS cells and iPS-CMs from the NVM patient and the healthy volunteer.Immunofluorescence assay showed that the iPS cells expressed pluripotent stem cell markers Oct4,Sox2,Ssea4 and TRA-1-81.iPS cells exhibited a high alkaline phosphatase activity with a normal karyotype,and could be induced to differentiate into 3 germ layers in vitro.Immunocytochemical staining revealed that iPS-CMs expressed cardiac myocyte-specific markers TNNT2 andα-actinin.Patch-clamp experiment demonstrated the presence of functional cardiac-specific voltage-gated Na^+currents and action potentials in the iPS-CMs.Whole-exome sequencing showed similar genetic profiles between the blood samples,iPS cells and iPS-CMs of the NVM patient,and several gene mutations were found to have potential associations with NVM.Conclusion We successfully obtain iPS cells and iPS-CMs from the NVM patient,and these cells provide useful cell models for the study of NVM using iPS technology.

关 键 词：心肌致密化不全 诱导多潜能干细胞 心肌样细胞 尿液细胞 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学]