目的:建立致心律失常性右室心肌病(ARVC)患者特异性的诱导性多能干细胞(iPSCs),为研究ARVC发病机制提供研究模型.方法:培养来源于ARVC患者皮肤成纤维细胞,并进行突变位点测序鉴定.通过仙台病毒转导入外源性多能转录因子,将ARVC患者皮肤细胞诱导为iPSCs,结合免疫荧光法,实时荧光定量PCR,以及体内外三胚层形成实验对iPS细胞全能型进行鉴定.通过调控Wnt信号通路诱导iPS细胞定向分化为心肌细胞.结果:ARVC患者来源的iPSCs显示碱性磷酸酶阳性,多能性相关基因高表达,胚胎干细胞标志物Oct4,SSEA4,TRA-1-81阳性.体外悬浮培养形成的拟胚体以及体内畸胎瘤形成实验均显示ARVC-iPSCs具有向3个胚层分化能力.经过体外心肌定向,ARVC-iPSC可诱导产生自主节律性搏动细胞团,免疫荧光显示cTnT阳性.结论:本研究使用仙台病毒,建立了无插入型ARVC患者特异的诱导性多能干细胞系,该细胞系具有多能分化特性,并可定向分化为心肌细胞,为研究ARVC的致病因素和药物筛选提供宝贵的实验模型.%Objective:To establish the induced pluripotent stem cells (iPSCs) from patients with arrhythmogenic right ventrcular cardiomyopathy (ARVC).Methods:The fibroblasts were isolated from ARVC patient and DNA mutation sites were confirmed.We reprogrammed the patient fibroblasts to pluripotency by sendaiviral transduction with defined pluripotency factors.Then,we evaluated the pluripotency of ARVC-iPSCs by performing the immunofluorescence analysis,real-time PCR and 3 germ layer formation assay.We also induced the ARVC-iP-SCs into cardiac specific differentiation by regulating Wnt signaling pathway.Results:Apart from alkaline phosphatase positive staining,iPSCs derived from ARVC patients expressed pluripotent related genes and embryonic stem cell marker OCT4,SSEA4 and TRA-1-81.Embryonic body formation in vitro and teratoma test in vivo demonstrated that ARVC-iPSCs could differentiate into all three germ layers.After in vitro cardiac differentiation,ARVC-iPSC could be successfully induced to spontaneously beating mass with cTNT staining positive.Conclusion:Induced pluripotent stem cell was successfully established by integration free sendai virus from dermal fibroblast of patients with ARVC.It would provide the valuable experimental model to study the molecular mechanism of ARVC.
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