Spraque-Dawley大鼠心室肌细胞分离和胞内钙离子浓度测定

Isolation and cytosolic calcium assay of ventricular myocytes of Spraque-Dawley rat

目的探讨Spraque-Dawley大鼠心室肌细胞分离和胞内钙离子浓度测定方法。方法采用三步法行逆行主动脉灌流获得单个耐钙心肌细胞,然后采用胞内钙荧光测定技术测定细胞内钙离子浓度。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。采用荧光探针Fura-2/AM,可准确测定细胞内钙离子浓度。结论通过本研究采用的方法,不仅可获得大量耐钙心肌细胞,而且可准确测定细胞内钙离子浓度。

Objective To investigate methods of isolating Spraque-Dawley rat ventricular myocytes and assaying cytosolic calcium. Methods Calcium-tolerant ventricular myocyte was obtained by three-step retrograde perfusation, and cytosolic calcium concentrations were assayed by fluorescent probe Furo-2/AM. Results If the whole heart remained red in the process of isolation, the numbers of total cell and calcium-tolerant cell were more than 90 percent and about 70 percent respectively. The numbers of total cell and calcium-tolerant cell in local red parts were more than 80 percent and about 60 percent respectively, however, there were a great varieties in numbers in pale parts, which were generally less than 50 percent and calcium-tolerant cells were less. ff the whole heart remained pale in the process of isolation, the numbers of total cell were less than 30 percent and almost no calcium-tolerant cells. Cytosolic calcium concentrations were accurately assayed by fluorescent probe Fura-2/AM.Conclusion If this method is adopted strictly in the process of isolation, Not only a large number of calcium-tolerant cells can he obtained, but also cytosolic calcium concentrations can be accurately assayed.