一种改进的人体心房肌细胞分离方法.pdf

858 生理学报 Acta Physiologica Sinica, December 25, 2007, 59 (6): 858-864 实验技术 一种改进的人体心房肌细胞分离方法 * 李妙龄，曾晓荣 ，杨艳，刘智飞，丁银元，周文，裴杰 泸州医学院心肌电生理学研究室，泸州 646000 摘 要：本研究旨在探讨稳定的人体心房肌细胞分离方法，并观察分离的正常窦性心律(normal sinus rhythm, NSR)患者右心 房肌细胞基本电生理特性。XXIV 型蛋白酶和V 型胶原酶两步法进行单个人体心房肌细胞分离，常规全细胞膜片钳技术记 录正常的 L- 型钙通道电流(L-type calcium current, ICa-L) 、钠通道电流(sodium current, INa ) 、瞬时外向钾通道电流(transient outward potassium current, Ito1)和内向整流钾通道电流(inward rectifier potassium current, IK1) 。此方法分离的人体心房肌细胞 数量多，细胞膜光滑，折光性强，横纹清楚，耐钙，可用于膜片钳实验的占分离细胞总数的 50 %~ 60 % 。该方法简单、 稳定、可靠，酶用量少，分离的心肌细胞质量好，数量多，并能记录到多种离子通道电流，表明其具有正常的电生理 功能，适合膜片钳实验。 关键词：心房肌；细胞分离；膜片钳；离子通道 中图分类号：R 3 2 9 An improved method for isolation of single atrial myocyte from human heart * LI Miao-Ling, ZENG Xiao-Rong , YANG Yan, LIU Zhi-Fei, DING Yin-Yuan, ZHOU Wen, PEI Jie Department of Myocardial Electrophysiology, Luzhou Medical College, Luzhou 646000, China Abstract: To approach the method of isolation of tolerant human atrial myocytes, single myocytes were isolated by modified procedure of enzymatic dissociation with protease (type XXIV) and collagenase (type V). L-type calcium channel current (ICa-L), sodium current (INa ), transient outward potassium current (Ito1 ), and inward rectifier potassium current (IK1) in isolated atrial myocytes were recorded by using whole-cell patch-clamp techniques. Single cardiocytes isolated by this method were smooth, well-striated and rod-shaped. The yields of recordable myocytes, which were viable and calcium-tolerant for electrophysiological studies, were 50%-60% of the total isolated cells. Compared with other isolation methods, this method was simple and steady, but with yield of a great number of qualified myocytes. The currents recorded in these cells were functional and active. Our research suggests