钾离子通道论文：针对HERG钾通道的早期心脏毒性评价的高通量筛选细胞模型.docVIP

钾离子通道论文：针对HERG钾通道的早期心脏毒性评价的高通量筛选细胞模型 【中文摘要】药物研发失败的重要原因之一是由于其心脏毒性。很多化合物由于非特异性的抑制了心肌细胞中的hERG (human ether-a-go-go-related gene)钾离子通道而导致心率失常,严重时则导致病人猝死。有些药物在研发后期或者已经上市之后,才发现其抑制hERG钾通道而导致心脏毒性,最终不得不撤出市场。因此建立针对hERG钾通道的心脏毒性评价体系在国际药物研发领域是必须的步骤,从而实现“早期发现早期淘汰”,为新药研发节省时间和费用。检测药物的hERG心脏毒性,目前最常用方法主要为传统的膜片钳技术。由于膜片钳技术对实验人员要求相对比较高,且通量低,仅适于候选药物的确定或新药申报阶段,及在检测少量化合物时方可用使用。因此发展针对hERG钾通道的高通量筛选模型是药物研发的必然趋势。而基于荧光的高通量筛选技术利用hERG通道对金属铊离子的通透性,又采用对铊离子敏感的荧光染料,配备全自动化荧光检测系统,可实现大批量化合物的筛选来检测药物对hERG通道的作用。此方法通量高、测试周期短,适于结构多样的化合物库的检测及先导化合物发现阶段大批量化合物hERG心脏毒性的早期初筛。目前在国内还没有成熟的针对hERG的高通量筛选模型,为了检验我们建立的hERG钾通道的高通量筛选方法,我们研用经典的全细胞膜片钳技术进行了对照。通过全细胞膜片钳技术记录在HEK (human embryonic kidney)293细胞上瞬时表达的hERG钾通道的电流和动力学曲线(激活、失活、复活和去活化)来研究几种不同化合物对hERG电流及动力学曲线的影响。同时建立稳定表达hERG基因的HEK293细胞株,采用铊离子荧光检测法对上述几种化合物进行检测,验证该方法的可行性及其与膜片钳技术的相关性。本论文建立了稳定评价hERG心脏毒性的基于荧光的高通量筛选系统,这将加快新药研发的进程,节省新药研发的费用,有重要的应用价值。 【英文摘要】Long QT syndrome can result in ventricular arrhythmia (torsade de pointes) and sudden death. hERG channel inhibition through drugs is now recognized as a common reason for the acquired form of long QT syndrome. So nowadays, hERG channel is becoming a hot research aspect all over the world. Many drugs have been withdrawn from the market because of the heart adverse reaction:long QT syndrome, due to their inhibition of hERG channel. Therefore Food and Drug Administration regulations now require the assessment of compound activity on the hERG channel. And it has become an important part of the safety evaluation in the process of new drug application.To detect compound effects on human ether-a?go-go related gene (hERG) potassium channels, In addition to radioactive ligand displacement tests, there are two other common approaches. The one is surrogate ion-based flux assays, the other is electrophysiological recordings. The former is much high-throughput, whereas the latter measure the effects on ion channel directly. Electropysiology has been thought the golden standard for detecting ion channel. But it is expensive, high-level, and