成年家兔心房肌细胞的分离和电生理记录

Isolation and electrophysiological recording of adult rabbit atrial myocytes

目的建立一种简单稳定的成年家兔心房肌细胞的分离、培养方法并进行电生理记录。方法麻醉后取出成年家兔心脏,采用Langendorff灌流装置及急性酶裂解法分离心房肌细胞,差速贴壁法进行纯化后培养于DMEM培养基。在倒置显微镜下观察细胞形态,利用透射电镜观察细胞超微结构,用免疫荧光染色法对心房肌细胞进行鉴定,利用全细胞膜片钳技术记录动作电位和内向钙电流和外向钾电流。结果本方法分离的心房肌细胞纯度和细胞存活率较高,并使用膜电钳技术成功记录了L-型钙电流和瞬时外向钾电流。结论该方法简便有效,细胞存活率高且为进一步进行各种电生理实验打下了基础。

AIM To construct a simple and stable method of isolating single atrial myoctes in adult rabbits for culturing and various electrophysiological recording. METHODS Single atrial myoctes of adult rabbits were isolated enzymatically by modified Langendorff perfusion technique and were cultured in DMEM culture media after purification. Morphological and ultrastracture were examined by inverted microscope and transmission electron microscope and immunofluoreseenet examination was used to identificate the atrial myocytes. Patch clamp was used to record the membrane currents, including ICa-L, and Ito current with whole-cell recording mode. RESULTS The survival rate and purity were satisfactory and L-type calcium current and transient outward potassium current were recoded in single atrial myocytes. CONCLUSION This method is simple and effective to get single adult atrial myocytes, which is can be used for further research on electrophysiological properties.