正常耐钙Spraque-Dawley大鼠心室肌细胞分离方法及体会

目的探讨稳定分离用于膜片钳实验的耐钙Spraque-Dawley大鼠心室肌细胞的方法。方法在自制心脏灌流装置上,采用三步法行逆行主动脉灌流获得单个耐钙心肌细胞,以膜片钳全细胞方式记录离子流。结果在分离过程中如整个心脏保持红润,则细胞数量在90%以上,耐钙细胞KB液中孵育后在70%左右;在分离过程中心脏局部保持红润部位的细胞数量在80%以上,耐钙细胞在60%左右,而苍白区细胞数量变异较大,但一般均在50%以下,且耐钙细胞较少;在分离过程中如整个心脏始终苍白,则细胞数量不仅低于30%,且几乎没有耐钙细胞。结论在心肌细胞分离过程中如严格按照本文的介绍,能获得大量具有正常电生理特性的耐钙心肌细胞。

Isolation method and experience of normal calcium-tolerant ventricular myocytes of Spraque-Dawley rat

Objective To investigate the method of stably isolating Spraque-Dawley rat calcium-tolerant ventricular myocytes for patch clamp techniques. Methods Calcium-tolerant ventricular myocyte was obtained by three-step retrograde perfusing with perfusing apparatus made by ourselves. Ion currents were be recorded by using myocytes after incubation in KB solution with whole cell configuration. Results If the whole heart remained red in the whole process of isolation, the numbers of total cell and calcium-tolerant cell were more than 90 percents and about 70 percents respectively. The numbers of total cell and calcium-tolerant cell in local red parts were more than 80 percents and about 60 percents respectively. However, there were a great varieties in numbers in pale parts, which were generally less than 50 percents and calcium-tolerant cells were less. If the whole heart remained pale in the process of isolation, the numbers of total cell were less than 30 percents and almost no calcium-tolerant cells. Conclusion If this method is adopted strictly in the process of isolation, a large number of calcium-tolerant cells with normal electrophysiology can be obtained.