乳鼠心肌细胞原代培养及电生理特征记录

Primary culture of neonatal rat ventricular myocytes and recording of their electrophysiological properties

目的分离培养原代乳鼠心室肌细胞,观察其搏动性,并探索其静息电位(RP)、自发性动作电位(AP)等电生理特征。方法用0.1%胰蛋白酶分离新生1～2 d SD大鼠心室肌细胞,并用差速贴壁法和5-溴脱氧尿嘧啶纯化心肌细胞;通过光镜观察心肌细胞形态和搏动情况;膜片钳技术记录心肌细胞的RP、自发性AP及钠离子电流(INa)特征。结果光镜下见培养12 h后心肌细胞基本贴壁,培养至第5 d时85%左右的心肌细胞都有自发性搏动,搏动频率80次/分左右;记录到自发性搏动心肌细胞RP为-85.34±4.15 mV,并可记录到同步产生的自发性AP,频率84±12次/分,而无自发搏动的心肌细胞RP为-42.1±6.5 mV,未能记录到自发性AP;心室肌细胞INa的阈电位为-60mV。结论该方法分离培养的原代乳鼠心室肌细胞搏动比例高,同时证实了自发搏动性是乳鼠原代心肌细胞状态佳,并具有良好电生理特征的重要标志。

Objective To explore the condition and approach to the primary cuhure of neonatal rat ventricular myocytes, and to record their electrophysiological characteristics. Methods The ventricles of 1 -2 days neonatal rats were digested with 0. 1% typsin. The cell suspension was preplated for 90 minutes to reduce the proportion of nonmyocardial cells. Cultures were treated for 6 days with bromodeoxyuridine （ BrdU）, 0. 1 mmol/L, to arrest the proliferation of nonmyocardial cells. Myocytes could be identified by light-electricity microscop. Then rest potential, spontaneous action potentials and INa of myocardial cells was recorded by the patch-clamp technique. Results The cells spread over the first day, developed their characteristic morphology. 85% myocytes beating at the frequency of about 80 beating per minutes（ bpm） in the fifth day. The rest potential was about - 85.34 ± 4.15 mV, and spontaneous action potentials outbreak with the mean rate of 84 ± 12 bpm in beating neonatal rat ventricular myocytes. Threshold of INa was - 60 mV in myocytes. Conclusion This technique for isolation of myocytes is effective in acquiring beating myocytes that bear the normal eletrophysiologicat characteristics.