二步酶消化法分离大鼠主动脉血管平滑肌细胞及活性鉴定

Isolation of Rat Aortic Vascular Smooth Muscle Cells with Two-step Digestion Method and Activity Identification

目的:建立一种有效、方便的大鼠血管平滑肌细胞(VSMCs)的酶解分离方法。方法:采用二步酶消化法分离VSM-Cs,显微镜下观察细胞形态,锥虫蓝染色观察细胞成活率,荧光显微镜检测细胞内钙荧光强度(FI)在KCl刺激下的变化,全细胞膜片钳记录电压-依赖性钾电流。结果:分离的VSMCs镜下呈典型的梭状或长条状,成活率达68.0%;VSMCs内钙FI在KCl刺激下可增高;并可记录到稳定的电压-依赖性钾电流。结论:本法可获得大量形态和结构良好的VSMCs,且胞膜离子通道功能良好。

Objective： To establish an effective and convenient enzya^lysis method to isolate rat vascular smooth mus- cle cells（VSMCs）. Methods： The two-step digestion method was used to isolate VSMCs. The cell forms were observed under the microscope, and the cell viability was detected with trypan blue stain. The change of cell calcium fluorescence intemity（H）was detected with the stimulation of potassium chloride（ KC1） under fluorescence microscope. The voltage de- pendence potassium current was recorded with whole cell patch-clamp technique. Results： The ：isolated VSMCs possessed the forms of typical spindle or long-strip and the cell viability was 68.0%. The calcium FI ot! VSMCs increased under KCI stimulation. The voltage dependence potassium current could be recorded steadily. Conclusion： A large number of VSMCs with good morphology and structure are obtained by this method. The ion channels of VSMC membrane possesses good reaction.