大鼠冠状动脉平滑肌细胞的分离及电压依赖性钾通道电流特性的初步研究

Isolation of rat coronary arterial smooth muscle cells and preliminary study of the characteristics of the voltage dependent potassium channel

目的建立一种稳定、高效的大鼠冠状动脉平滑肌细胞(CASMCs)的急性酶分离方法,并对其电压依赖性钾离子通道(KV)进行研究。方法采用"三步"(依次用3种分离酶液)急性酶分离方法分离大鼠CASMCs,并进行细胞鉴定,采用膜片钳记录KV电流。结果①本分离方法可以获得数量较多,活性较好的平滑肌细胞;②测试电位为+60 mV时,KV电流密度达到最大值34.35±2.53 pA/pF,加入4-氨基吡啶后电流密度降至3.55±0.47pA/pF。两组之间电流密度的差异具有显著性(n=5,P<0.05)。KV稳态激活曲线的半数激活电压V1/2=23.66±1.03 mV,曲线斜率因子k=14.44±0.94 mV。结论 "三步"法为使用膜片钳记录CASMCs的离子通道电流提供了较好的研究方法。

Objective To establish a stable and efficient enzyme separation method to isolation ot rat coronary artery smooth muscle cells （ CASMCs）, and to study on the voltage dependent potassium ion channels （ Kv ）. Methods CASMCs were isolated by a ＂three-step＂ acute enzyme separation method and identified by immunofluorescence. Kv currents were recorded by the patch-clamp technique. Results ①An abundance of CAMSCs with good reactivity were obtained by this method. ②At the test potential of ＋60 mV, the Kv current average current densities were 34.35±2.53 pA/ pF （n = 5 ）. After added 4-AP, the Kv current average current densities were 3.55±0.47 pA/pF at the same test potential （ P〈 0.05）. The half maximal activation voltage was 23.66±1.03 mV, and the slope value （k） was 14.44±0.94 mV. Conclusions ： This method can acquire a large number of CASMCs which can be used on the study of ion channels.