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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。
Objective To establish a simple and stable adult rabbit atrial myocyte isolation and culture methods and electrophysiological records. Methods The heart of adult rabbits was removed after anesthesia. The atrial myocytes were isolated by Langendorff perfusion and acute enzymatic cleavage. The cells were purified by differential adherence and cultured in DMEM medium. The morphology of the cells was observed under an inverted microscope. The ultrastructure of the cells was observed by transmission electron microscopy. The atrial myocytes were identified by immunofluorescence staining. The whole-cell patch-clamp technique was used to record the action potentials and inward and outward potassium current. Results The purity of atrial myocytes and the viability of cells isolated by this method were high. L-type calcium current and transient outward potassium current were recorded successfully by using membrane clamp technique. Conclusion The method is simple and effective, high cell survival rate and lay the foundation for further electrophysiological experiments.