通过iPS细胞模型对HERG基因突变L539fs/47导致长QT综合征的机制及药物干预研究

Induced pluripotent stem cells(iPS cells) are a type of pluripotent stem cell artificially derived from the adult somatic cells by reprogramming the cells into a pluripotent state.iPS cells can differentiate into myocytes with cardiac-specific molecular, structural, and functional properties. These results, coupled with the potential of this technology to generate patient-specific iPS lines, hold great promise. Long QT syndromes are heritable diseases associated with prolongation of the QT interval on an electrocardiogram. In long-QT syndrome type 2, mutations occur in the HERG gene.We have screened a family affected by long-QT syndrome type 2 and identified autosomal dominant large fragment deletions mutation (L539fs/47) in the HERG gene. We are to obtaine dermal fibroblasts from two family members and two healthy controls and infect them with retroviral vectors encoding the human transcription factors(SOX-2, KLF-4和 OCT-4 )to generate pluripotent stem cells, which then will be directed to differentiate into cardiac-like myocytes according to a specific protocol. With the use of immunohistochemistry, gene analyze, whole cell patch clamp and Micro-motor matrix record, cardiac-like myocytes are being characterized to show the disease's characteristics, establishing a convenient and powerful system for studying mechanisms of pathogenesis and provide experiment evidence for clinic through the drug intervention.

诱导性多功能干细胞（iPS细胞）是诱导成体细胞重新编码生成的一类多潜能干细胞。iPS细胞技术在心血管医学研究领域具有广阔的应用前景。iPS细胞可以分化成具有心肌特异性分子标志、结构和功能的细胞。先天性长QT综合症（LQTS）是一类由于QT间期延长导致的遗传性心脏病，其中HERG基因发生突变与2型LQTS密切相关。本实验室前期通过对一LQT2家系进行基因突变位点的筛选，发现了一个新的HERG基因突变（L539fs/47）。本研究拟通过转录因子组合(SOX-2, KLF-4和OCT-4)对来自该家系正常人与患者的皮肤纤维母细胞进行诱导，生成iPS细胞，并引导其分化成心肌样细胞，继而利用免疫组化、基因表达分析、全细胞膜片钳记录、微电极矩阵记录等方法，对心肌样细胞的特征进行研究，阐明该突变导致LQTS的发病机制,并通过药物干预实验为临床治疗提供实验依据。

由于携带突变(L539fs/47)的 LQT2 患者不幸猝死，未能获取该患者的皮肤纤维母细胞建立L539fs/47突变的iPSC模型进行L539fs/47致病机制的研究和药物干预实验。因此，本项目调整实验方案，建立异源表达系统模型进行L539fs/47致病机制和药物干预实验。研究发现，（1）L539fs/47-hERG 导致hERG通道蛋白表达降低，发生转运障碍；L539fs/47对hERG通道具有显著的负显性抑制作用，这可能是L539fs/47-hERG携带者发生恶性心律失常、心源性猝死的风险增加的原因。（2）L539fs/47使hERG通道对索他洛尔的敏感性增加，这可能是L539fs/47-LQT2患者较一般人群在使用抗心律失常药物时易发生药物性LQTS及TdP的原因，为临床安全用药提供指导。（3）庆大霉素可以改善杂合L539fs/47-hERG通道的表达和功能，为临床针对L539fs/47-LQT2患者制定治疗策略提供实验依据。

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