一种改进的人体心房肌细胞分离方法

An improved method for isolation of single atrial myocyte from human heart

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,I_(Ca-L)、钠通道电流(sodium current,I_(Na)、瞬时外向钾通道电流(transient outward potassium current,I_(to1)和内向整流钾通道电流(inward rectifier potassium current,I_(K1)。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%～605。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。

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 （Itol）, 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 that the myocytes isolated by the described method in this paper have normal electrophysiological function and are appropriate for patch-clamp experiments.