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

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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 40n th generation.n Conclusion:A patient specific BrS-iPSCs with pluripotent, normal karyotype and no exogenous gene insertion was successfully established and identified in this study.
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