摘要
目的:通过诱导多潜能干细胞(induced pluripotent stem cells,iPSC)体外定向分化心肌细胞(iPSC derived cardiomyocytes,iPSC-CM)的方法建立酒精性心肌病的电生理研究模型。方法:体外扩增培养人源iPSC并定向诱导其分化为iPSC-CM。实验将iPSC-CM分为空白对照组、酒精实验组和KN93干预组。通过免疫荧光法检测iPSC-CM的分化情况,采用细胞计数试剂盒(cell counting kit-8,CCK8)和乳酸脱氢酶(lactate dehydrogenase,LDH)活力检测试剂盒检测iPSC-CM的功能,使用实时无标记细胞分析仪(real time cellular analysis,RTCA)监测iPSC-CM电活动的变化,采用线粒体膜电位荧光探针JC-1染色联合RTCA分析仪观察酒精对iPSC-CM的损伤而进一步验证模型。结果:与空白对照组(0 mg/mL酒精)相比,不同剂量(25,50,100,150,200,250,300 mmol/L)的酒精实验组能抑制iPSCCM的增殖,并呈剂量依赖性,差异均有统计学意义(均P<0.05);与空白对照组(0 mg/mL酒精)相比,浓度为100 mmol/L的酒精实验组中iPSC-CM出现明确的损伤,LDH释放量增加,线粒体膜电位下降,其震动幅度和跳动频率亦下降,差异均有统计学意义(均P<0.05);与浓度为100 mg/mL的酒精实验组相比,KN93干预组可通过KN93阻断坏死性凋亡通路而缓解酒精对iPSC-CM的损伤,LDH的释放量减少,线粒体膜电位上升,其震动幅度和跳动频率亦上升,差异均有统计学意义(均P<0.05)。结论:基于体外心肌分化的方法可成功建立酒精性心肌病电生理研究模型,该模型可用于研究体外心肌电生理活动和相关疾病的分子机制,可为药物筛选、疾病机制研究和临床实验提供更加合理而高效的研究工具。
Objective:To establish an electrophysiological model of alcoholic cardiomyopathy by inducing pluripotent stem cells(iPSCs)to differentiate into cardiomyocytes(iPSC-CM)in vitro.Methods:The human iPSC were expanded in vitro and differentiated into iPSC-CM.The iPSC-CM were divided into a blank control group,an alcoholic experiment group(according to the concentration of alcoholic,the alcoholic experiment was also divided into many subgroups),and a KN93 treatment group.Then the efficiency of iPSC differentiated to iPSC-CM was detected by immunofluorescence,the function of iPSC-CM was detected by cell counting kit-8(CCK8)assay and lactate dehydrogenase(LDH)activity assay kit.The electrophysiological activity of iPSC-CM was monitored by real time cellular analysis(RTCA),the injury of iPSC-CM caused by alcohol was further verified by the mitochondrial membrane potential fluorescence probe JC-1 staining combined with RTCA analysis.Results:Compared with the blank control group,the different doses(25,50,100,150,200,250,300 mmol/L)of alcohol could significantly inhibit the proliferation of iPSC-CM in a dose-dependent manner(all P<0.05).Compared with the blank control group,the activity of iPSC-CM was significantly reduced by 100 mmol/L alcohol,resulting in the increase of LDH release,the decrease of mitochondrial membrane potential,the amplitude and beating rate(all P<0.05).Compared with the 100 mg/mL alcoholic experiment group,the KN93 treatment group significantly alleviated the damage of alcohol to iPSC-CM by blocking the necrotic apoptotic pathway,resulting in the decrease of LDH release,the increase of mitochondrial membrane potential,the amplitude and beating rate(all P<0.05).Conclusion:The electrophysiological model of alcoholic cardiomyopathy based on the differentiation of cardiomyocytes are successfully established,which can be used to study the electrophysiological activity and the molecular mechanism for relevant diseases,and it may provide a more reasonable and effective research tool for drug screening and clinical study.
作者
李弘夏
黄美园
王金文
陈栋良
LI Hongxia;HUANG Meiyuan;WANG Jinwen;CHEN Dongliang(Department of Pathology,Affiliated Zhuzhou Hospital,Xiangya School of Medicine,Central South University,Zhuzhou Hunan 412007,China)
出处
《中南大学学报（医学版）》
CAS
CSCD
北大核心
2020年第4期386-394,共9页
Journal of Central South University ：Medical Science
基金
湖南省卫生与计划生育委员会课题(B20180254)。
关键词
干细胞
诱导多潜能干细胞
心肌细胞
酒精性心肌病
电生理学
stem cell
induced pluripotent stem cells
cardiomyocytes
alcoholic cardiomyopathy
electrophysiology
