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目的:建立高效可靠的成年犬心肌细胞分离方法,获得高产量与高质量的心肌细胞,以便进行犬心肌细胞收缩功能的研究。方法:采用改良的Langendorff灌流胶原酶消化法分离得到左心室心肌细胞。荧光显微镜观察细胞生长状态和形态并利用单细胞收缩动态边缘检测系统测定心肌细胞收缩功能的改变。结果:结果显示,与传统方法相比,即刻分离的心肌细胞状态良好,复钙后的心肌细胞成活率达到70%-80%,转染重组腺病毒β2-EGFP培养48 h,心肌细胞成活率为60%-70%。给予持续电场刺激,心肌细胞可以保持收缩节律和幅度稳定30 min以上。结论:该方法操作简单,分离的活细胞产量高,质量好,节约实验成本和时间,为心血管相关研究提供良好的细胞模型。
OBJECTIVE: To establish an efficient and reliable method for the isolation of cardiomyocytes from adult canine and to obtain cardiomyocytes of high yield and quality for the study of canine cardiomyocyte contractile function. Methods: Left ventricular myocytes were isolated by modified Langendorff perfusion collagenase digestion. Fluorescence microscopy was used to observe the cell growth status and morphological changes and the changes of contractile function of cardiomyocytes were detected by single-cell contraction dynamic edge detection system. Results: Compared with the traditional method, the isolated cardiomyocytes were in good condition and the viability of cardiomyocytes after reuptake was 70% -80%. The survival rate of cardiomyocytes was 48 h after transfection with recombinant adenovirus β2-EGFP Is 60% -70%. Given sustained electric field stimulation, cardiomyocytes can maintain a steady rhythm of contractions and amplitude stability for more than 30 min. Conclusion: The method is easy to operate, the isolated living cells have high yield, good quality, saving experimental cost and time, and provide a good cell model for cardiovascular related research.