小鼠心室肌细胞分离方法的改进

Improvement of the method to separate cardiac ventricular myocytes from mice

目的:探索更简单实用和重复性好的小鼠心肌细胞分离技术。方法:实验于2005-05/09在大连大学医学院医学研究中心完成。选择8～16周龄昆明小鼠150只,雌雄不拘,体质量30～40g,由大连大学医学院动物中心提供。采用原位主动脉插管和动脉夹固定法酶解分离小鼠心室肌细胞,并在室温下进行全细胞膜片钳记录。结果:可将主动脉插管的时间控制在1.5min内,所获心室肌细胞横纹清晰,一次性复钙可获得50%～60%的耐钙细胞,分离所得细胞可在全细胞膜片钳方式下记录到一过性外向钾电流(Ito)。结论:原位主动脉插管和动脉夹固定酶解分离心室肌细胞的方法简便易行,重复性好,克服了原有方法体外心脏插管时间长,每次插管时间差异较大,分离效果不稳定的弊端。

AIM： To explore an easier, more practical and reliable technique for the isolation of cardiac ventficular myecytes from mice. METHODS： The experiment was conducted in the Institute of Basic Medical Sciences, Medical College of Dalian University between May and September 2005. A total of 150 Kunming mice, male or female, at 8-16 weeks old with 30-40 g body mass were selected （provided by Animal Center of Medical College of Dalian University）. The cardiac ventricular myecytes were digested with enzyme and isolated by in situ aortic cannula and arterial fixation with a clip, so as to conduct the whole-cell path clamp recording in the room temperature. RESULTS： By this method, the cannula could be done within 1.5 minutes to obtain cardiac ventricular myecytes with clear cross-striation, and 50%- 60% calcium-resistant cardiac ventricular myocytes could be obtained after disposable recalcifiation. The transient outward potassium current Ito was successfully recorded by whole-cell patch clamp recording. CONCLUSION： This improved method is more convenient and reliable compared with the old one, by using which, it takes more time in cannula, there are obvious differences in cannula time, and the isolated effect is not stable.