基于人诱导性多能干细胞技术的长QT综合征III型发病机制及药物筛查研究

Inherited long QT syndromes (LQTs), characterized by electrocardiographic (ECG) QT interval prolongation and episodes of ventricular tachycardia and fibrillation, are major causes of sudden cardiac death in children and young adults. Thirteen genes are associated with LQTS. SCN5A encoding cardiac sodium channels is the causative gene underlying LQT type 3 (LQT3). The consequences of SCN5A mutations are patient-specific. Different mutation positions and functional features produce a phenotypic spectrum ranging from being asymptomatic to causing potentially fatal arrhythmias. Development of disease cell models specific to the patient's mutation leading to personalized treatment is a current focus of research. The present proposal is based on a recently reported LQT3-related SCN5A-A1180V mutation. It will use somatic reprogramming techniques to develop LQT3 patient SCN5A-A1180V mutation-specific induced pluripotent stem cell (iPSC) by introducing Oct3/4,Sox2, LANOG and LIN28 into patient dermal fibroblasts. iPSCs are further differentiated into cardiomyocytes (iPSC-CM) by co-culturing iPSCs with EDN-2. The electrophysiological characteristics and expression profiles of SCN5A-A1180V-iPSC-CM cell models including action potential, ion current and gene/protein expression characteristics will be tested using patch clamp, laser focus microscopy, and molecular biology techniques. Drug screen investigation will be performed using high throughput patch clamp and multi electrode arrays to identify shortened or prolonged QT interval and the actions of candidate drugs. Computational modeling will be developed using the electrophysiological and biological results. The proposal would benefit the future development of personalized medicine in the management of inherited arrhythmia patients.

遗传性长QT综合征III型（LQT3）的致病基因SCN5A编码心脏钠通道， 建立患者突变特异性体外细胞模型是实现个性化医疗的关键。本研究在前期基因测序确定一个LQT3家系SCN5A-A1180V突变基础上，通过体细胞重编程，将Oct3/4、Sox2、LANOG 和 LIN28四种转录因子基因克隆入慢病毒载体，引入患者体细胞，通过生物学鉴定，建立LQT3患者SCN5A-A1180V突变特异性诱导多能干细胞(iPSC)细胞系；采用心肌细胞定向分化技术，将iPSC分化为功能性心肌细胞（iPSC-CM），建立LQT3患者突变特异体外细胞模型；运用膜片钳、激光共聚焦、分子生物学、多导电极阵列（MEA）技术研究SCN5A-A1180V-iPSC-CM心律失常发病机制，进行抗心律失常药物筛选，发现延长和缩短患者心肌细胞动作电位的药物，建立数学仿真计算机模型，探索遗传性心律失常个性化治疗新途径。

遗传性长QT综合征III型（LQT3）的致病基因SCN5A编码心脏钠通道， 建立患者突变特异性体外细胞模型是实现个性化医疗的关键。本研究在前期基因测序确定一个LQT3家系SCN5A-A1180V突变基础上，通过体细胞重编程，将Oct3/4、Sox2、LANOG 和 LIN28四种转录因子基因克隆入慢病毒载体，引入患者尿液上皮细胞，诱导分化呈iPSC,经过碱性磷酸酶活性检测，经过干细胞marker，SSEA3、SSEA4、TRA-1-60、TRA-1-81和Nanog免疫染色，iPSC完全表达干性markers，并进行iPSC支原体污染检测，确定iPSC没有支原体污染。将获得的iPSC与患者自身母上皮细胞进行STR比对，证实建立的iPSC和患者自身上皮细胞同源，三胚层分化实验确定建立的iPSC具有三胚层分化潜能。对建立的患者特异性iPSC进行突变位点SCN5A-A1180V突变位点Sanger验证，确定iPSC携带SCN5A-A1180V突变位点，经过上述验证，建立了建立了LQT3患者SCN5A-A1180V突变特异性诱导多能干细胞(iPSC)细胞系。采用心肌细胞定向分化技术，将iPSC分化为功能性心肌细胞SCN5A-A1180V-iPSC-CM，流式细胞术检测表达cTNT的iPSC-CM为93.49%。经过心肌细胞标记物ɑ-Actinin和cTnT免疫染色，SCN5A-A1180V-iPSC-CM表达ɑ-Actinin和cTnT，对SCN5A-A1180V-iPSC-CM进行突变位点SCN5A-A1180V突变位点Sanger验证，确定SCN5A-A1180V-iPSC-CM携带SCN5A-A1180V，确定建立LQT3患者突变特异体外SCN5A-A1180V-iPSC-CM细胞模型；膜片钳实验确定了SCN5A-A1180V-iPSC-CM动作电位明显延长(534±12 vs 345±25, n=8, P<0.01, 2Hz)动作电位（AP），证实SCN5A-A1180V-iPSC-CM的LQT表型，异丙肾上腺素明显缩短SCN5A-A1180V-iPSC-CM（534±30 vs 395±39，n=8, P<0.001）和对照组AP(444±30 vs 383±29, n=8, P<0.01),心得安没有明显缩短SCN5A-A1180V-iPSC-CM AP。