基于实时细胞分析技术的人诱导多能干细胞源性心肌细胞的药物毒性评价研究

Establishment of a cardiotoxicity drug screening model based on human induced pluripotent stem cell-derived cardiomyocytes

目的以人诱导多能干细胞来源心肌细胞(hiPSC-CMs)为模型,结合实时细胞分析(RTCA)技术建立并验证体外药物心脏毒性评估系统。方法选用不同浓度的胺碘酮、维拉帕米及多柔比星(阿霉素),分别与商业化来源的hiPSC-CMs孵育,利用RTCA系统实时观察药物作用时及洗脱后不同时间段hiPSC-CMs自主搏动(搏动频率和幅度)、细胞指数及校正的场电位时限(FPDc)变化。结果1μmol/L胺碘酮作用12 h后,hiPSC-CMs大部分搏动振幅被抑制;10μmol/L浓度胺碘酮作用48 h使细胞指数下降12.22%(P<0.05),自主搏动抑制(P<0.001),药物洗脱后未见自主搏动恢复。与对照组相比,100 nmol/L维拉帕米作用12 h使搏动振幅下降47.38%(1.05±0.08对0.55±0.13,P<0.001),FPDc缩短33.33%(P<0.05),在药物洗脱后该组搏动振幅、FPDc恢复;而10μmol/L维拉帕米作用12 h后搏动振幅完全被抑制,细胞指数下降9.60%(P<0.01),药物洗脱后未见恢复。多柔比星10 nmol/L、100 nmol/L组对各细胞参数无影响,而10μmol/L组作用12 h后细胞指数下降35.87%(P<0.001),自主搏动、FPDc受到抑制(P<0.001),洗脱后自主搏动未恢复。结论以hiPSC-CMs为模型,结合RTCA技术可在体外有效评估不同药物的药理学及毒性,为后续药物早期心脏药理学研究提供方法学参考和理论依据。

Objective To establish and validate a cardiac drug screening system in vitro by using human induced pluripotent stem cell-derived cardiomyocytes(hiPSC-CMs)and real-time cell analysis(RTCA)system.Methods Different concentrations of amiodarone,verapamil and doxorubicin were used to incubate with commercial hiPSC-CMs,respectively.The changes of spontaneous pulse(pulse frequency and pulse amplitude),cell index and corrected field potential duration(FPDc)of hiPSC-CMs were measured by RTCA system during drug treatment and after elution in different time periods.Results Most of the pulse amplitudes of hiPSC-CMs were inhibited by amiodarone(1μmol/L).Amiodarone(10μmol/L)induced a 12.22%reduction of cell index after 48 h postdosing(P<0.05),and it suppressed spontaneous activity(P<0.001)of hiPSC-CMs with no spontaneous activity recovery after drug washout.FPDc was shortened by 33.33%(P<0.05),and pulse amplitude decreased by 47.38%(1.05±0.08 vs.0.55±0.13,P<0.001)after 12 h disposal of verapamil at 100 nmol/L,the pulsatile amplitude and FPDc were recovered after drug washout.Verapamil(10μmol/L)suppressed spontaneous activity(P<0.001)with no spontaneous activity recovery after washout,which corresponded to a 9.60%reduction in cell index after 12 h treatment(P<0.01).Spontaneous activity,cell index and FPDc were unperturbed by 10 nmol/L and 100 nmol/L doxorubicin.However,spontaneous activity and FPDc were completely suppressed(P<0.001)by 10μmol/L doxorubicin with no recovery after drug washout.The suppression in spontaneous activity and FPDc were coincident with a 35.87%reduction(P<0.001)in cell index 12 h postdosing at 10μmol/L.Conclusion hiPSC-CMs combined with RTCA system can be used to evaluate the pharmacology and toxicity of drugs in vitro,which can be used for drug screening including various antiarrhythmic drugs in the early stage of drug development.