成年小鼠心房肌细胞分离方法的改进及钾电流记录

目的建立稳定、重复性好的小鼠心房肌细胞分离技术,观察小鼠心房肌细胞形态以及钾通道电流特性,为进一步研究小鼠心房肌细胞的电生理特性打下基础。方法在体视显微镜下行主动脉插管,应用Langendorff装置恒流灌流,依次用无钙台氏液、酶消化液(含Ⅱ型胶原酶和胰蛋白酶)灌流,灌流完成后,剪除心室组织,心房组织块用消化酶液(仅含Ⅱ型胶原酶)行二步消化,分离出小鼠单个心房肌细胞,采用标准的全细胞膜片钳技术记录动作电位和钾电流,电流的电压-电流曲线(I-V曲线)以电压为横坐标,每个电压值下的电流密度(pA/pF)为纵坐标拟合而成。结果本法分离所得小鼠心房肌细胞呈长梭形、横纹清晰,细胞耐钙,具有正常电生理活性,细胞贴壁好,从而易于形成高阻封接;记录小鼠心房肌细胞动作电位为近似于三角形,静息电位为(-70.2±4.9)mV;记录的瞬时外向钾电流(Ito)激活非常快,达峰值后快速失活,峰电流存在明显的电压依赖性和外向整流特征,电流峰值为(11.1±1.9)pA/pF;超速激活的延迟整流钾电流(IKur)激活迅速,几乎无延迟现象,失活缓慢,峰电流呈现出外向整流特征,该电流约在-20 mV被激活,峰电流存在明显的电压依赖性,峰值(7.0±1.5)pA/pF。内向整流钾电流(IK1)随着超极化刺激电位的增加,电流密度增加,在-140 mV时,其内向电流的峰值为(27.0±2.3)pA/pF。结论此方法可成功获取适合做膜片钳实验的单个小鼠心房肌细胞,操作方法简便,可行性高,重复性好。

Improved method for adult mouse atrial myocardium isolation and potassium currents recording

Objective To improve the method used to isolate adult mouse atrial myocytes and observe the characteristics of action potential（AP） and potassium currents （K^＋currents） of atrial myocytes. Methods Atrial myocytes from atrial myocardium of mouse on an in vitro Langendorff perfusion system were obtained by double enzymes digestion （collagenase type Ⅱ and trypsin） method. Whole-cell patch-clamp recording technique was used to record AP and K^＋currents. The current-voltage （I-V） curves of K^＋currents of atrial myocytes were fitted. Results Clear transverse striation could be observed in the cells of mouse atrial myocardium which were separated by this way. These cells also had normal electrophysiology activity. Giga-ohm seal could be easily achieved. The sharp of mouse atrial myocytes AP was triangles, and resting potential was （-70.2±4.9）mV;transient outward potassium current （Ito） activation was very fast, fast inactivation after the peak, the peak current was obvious and outwardly rectifying voltage-dependent characteristics, peak current density was （11.1±1.9）pA/pF;ultra-rapid delayed rectifier potassium current （IKur） activated rapidly, almost no lag, slow deactivation, go to the rectifier peak current rendering features, this current was activated at about -20 mV, the peak current was present apparent＆amp;nbsp;voltage dependence, peak current density was （7.0±1.5）pA/pF. Inward rectifier potassium current （IK1） as stimuli hyperpolarized potentials increases, the current density increases, at -140 mV, its inward peak current density was （27.0±2.3）pA/pF. Conclusion The way to isolate mouse atrial myocytes in this experiment is very convenient, and the cells not only are very easy to seal, but also have normal electrophysiology activity.