携带SCN3B-C.260突变的Brugada综合征患者特异性诱导多能干细胞的建立及鉴定  

Establishment and identification of induced pluripotent stem cells from patients with Brugada syndrome with SCN3B-C.260 mutation

作　　者：蒋华[1] 热米娜·阿卜杜热西提 热甫开提·阿不都哈力克 王坤[1] 吕文魁[1] 艾力曼·马合木提[1] Jiang Hua;Remina·Abudurexiti;Refukaiti·Abuduhalike;Wang Kun;Lyu Wenkui;Ailiman·Mahemuti(Heart Center,First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Department of Functional Examination,the Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学第一附属医院心脏中心,乌鲁木齐830054 [2]新疆医科大学第五附属医院功能检查科,乌鲁木齐830011

出　　处：《中华心力衰竭和心肌病杂志（中英文）》2020年第4期267-271,共5页Chinese Journal of Heart Failure and Cardiomyopathy

基　　金：国家自然科学基金项目(81660055)。

摘　　要：目的构建Brugada综合征(BrS)患者特异性诱导多能干细胞(iPSCs)模型并进行鉴定。方法利用一例携带SCN3B-C.260突变的女性BrS患者的外周血分离培养外周血单核细胞(PBMCs),通过仙台病毒,将转录因子Oct4、Sox2、Klf4和cMyc(OSKM)转染到PBMCs,采用非整合重编程技术构建特异性BrS-iPSCs。通过多能性相关基因表达产物(Oct4)免疫荧光染色检测、体外拟胚体形成及三胚层分化能力检测鉴定BrS-iPSCs的多能性,通过核型分析及仙台病毒特异的核糖核酸(RNA)检测鉴定BrS-iPSCs的安全性。结果构建的特异性BrS-iPSCs为典型的胚胎干细胞样克隆。免疫荧光染色显示多能性相关基因表达产物(Oct4)阳性,体外拟胚体形成及三胚层分化能力检测显示内胚层(甲胎蛋白)、中胚层(Brachyury)和外胚层(PAX-6)特异标记物免疫荧光染色呈阳性。G显带法染色体核型分析显示BrS-iPSCs染色体核型正常,为46XX,各染色体形态及区段无肉眼可见异常。对BrS-iPSCs进行仙台病毒基因组RNA的检测,在第40代时无法检测到仙台病毒基因组的存在。结论本研究成功构建了具有多能性、核型正常且无外源性基因插入的特异性BrS-iPSCs。Objective To establish and identify specific induced pluripotent stem cells(iPSCs)for a patient with Brugada syndrome(BrS).Methods Peripheral blood mononuclear cells(PBMCs)were isolated and cultured from the peripheral blood of a female patient with BrS.The transcription factors Oct4,Sox2,Klf4 and cMyc(OSKM)were transfected into PBMCs by use of Sendai virus and the specific BrS-iPSCs was established by non-integrating reprogramming technique.The pluripotency of BrS-iPSCs was identified by immunofluorescence staining of pluripotent associated gene expression product(Oct4),in vitro embryoid body(EB)formation and triembryo differentiation ability test.The safety of BrS-iPSCs was identified by karyotype analysis with Giemsa staining and Sendai virus specific RNA test.Results The specific BrS-iPSCs was typical embryonic stem cell-like clone.Immunofluorescence staining showed positive expression of pluripotent associated gene products(Oct4),and in vitro EB formation and triembryo differentiation ability tests showed positive immunofluorescence staining of endoderm(AFP),mesoderm(Brachyury)and ectoderm(PAX-6)specific markers.The karyotype analysis with Giemsa staining showed the karyotype of BrS-iPSCs was normal of 46XX with no macroscopic abnormalities in the morphology and segments of each chromosome.Sendai virus specific RNA could not be detected in the BrS-iPSCs in the 40th generation.Conclusion A patient specific BrS-iPSCs with pluripotent,normal karyotype and no exogenous gene insertion was successfully established and identified in this study.

关 键 词：BRUGADA综合征 外周血单核细胞 诱导多能干细胞 

分 类 号：R541.7[医药卫生—心血管疾病]