基于人胚胎干细胞的实时细胞系统评价心脏毒性方法的建立  

Establishment of the method to evaluate cardiac toxicity by real-time cell analysis system on human embryonic stem cells

作　　者：赵琪[1] 汪溪洁[1] 王淑颜 马璟[1] 

机构地区：[1]中国医药工业研究总院上海医药工业研究院国家上海新药安全评价研究中心,上海201203

出　　处：《中国药理学通报》2016年第1期138-143,共6页Chinese Pharmacological Bulletin

基　　金："十二五"国家科技部重大新药创制重大专项资助项目(No 2012ZX09302002)

摘　　要：目的采用人胚胎干细胞诱导分化的心肌细胞(human embryonic stem cells derived cardiomyocytes,h ESC-CM)联合实时细胞分析系统(real-time cell analysis Cardio,RTCA Cardio),建立一种体外药物心脏毒性早期筛选方法。方法将h ESC-CM接种到RTCA Cardio E-Plate 96孔板,分析心肌细胞的搏动频率、幅度和搏动节律不规则性指数确定细胞最佳接种密度及检测时间,建立体外药物心脏毒性早期筛选方法。在此基础上,分别采用0.1%DMSO作为溶剂对照及具有抗心律失常作用的药物奎尼丁(0.2、0.78、3.13、12.5、50和100μmol·L-1)作为阳性对照进行了验证。结果0.1%DMSO对h ESC-CM的生长指数(cell index,CI值)、搏动特征图谱及搏动频率和幅度均无影响。而与溶剂对照组相比,奎尼丁浓度≥3.13μmol·L-1时影响细胞CI值和特征性搏动图谱,抑制细胞搏动频率和幅度,且具有浓度依赖性,浓度越高细胞搏动信号恢复所需时间越长,对搏动频率和幅度的抑制作用越明显。结论采用h ESC-CM联合RTCA Cardio系统建立的体外药物心脏毒性早期筛选方法可以检测出奎尼丁对心肌细胞搏动状况的影响,该方法可作为一种高通量筛选工具用于临床前药物心脏毒性早期筛选。Aim To establish an in vitro early drug cardiac toxicity evaluation method by human embryonic stem cells derived cardiomyocytes( hE SC-CM) and real-time cell analysis Cardio( RTCA Cardio) system. Method The hE SC-CM were cultured at RTCA Cardio E-Plate 96. Impedance signals from hE SC-CM were analyzed for beating rate,contraction amplitude and beating rhythm irregularity to determine the optimum inoculation density and detection duration. Based on this,we used 0. 1 % DMSO to be the solvent and quinidine( 0. 2,0. 78,3. 13,12. 5,50 and100 μmol·L- 1) known as affecting cardiac activity to validate this method. Result The results revealed no significant changes in the cell index( CI),transient pulse patterns,beating rate and amplitude of hE SC-CM. Quinidine will affect the CI and transi-ent pulse patterns of hE SC-CM and decrease the beating rate and amplitude of hE SC-CM when its concentration ≥ 3. 13 μmol ·L- 1. And this effect is concentration-dependent,the higher the concentration,the more time they need to recover beating and the more significant the beating rate and amplitude inhibition of quinidine on hE SC-CM. Conclusion The method established by hE SC-CM and RTCA Cardio system can detect the effect of quinidine on the contraction of hE SC-CM,and this indicates that this method has the potential to be an attractive high-throughput tool for screening potential drugs in early evaluation of drug cardiotoxicity.

关 键 词：hESC-CM RTCACardio 心脏毒性 药物 体外筛选 奎尼丁 

分 类 号：R322.11[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学]