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目的:建立简便有效的心肌细胞分离和原代培养方法,获得具有理想数量和活性的原代心肌细胞。方法:无菌条件下取出生72h内的新生SD大鼠心脏,去除心肌结缔组织和心房组织,只保留左心室,采用胰酶和胶原酶Ⅰ联合消化、差速贴壁分离的方法,获得心肌细胞进行体外培养,观察细胞的形态变化,对细胞的搏动频率、搏动细胞数进行统计分析。结果:分离的原代心肌细胞生长良好,核仁清晰可见。高倍镜下观察,心肌细胞接种12h后,少数细胞贴壁生长,且贴壁的少数单个细胞出现自发搏动,之后搏动细胞数以及细胞搏动频率都逐渐增加,接种48h后细胞完全铺展开来并连接成片,自发搏动呈现同步性和岛屿状。结论:本实验方法获得的新生SD大鼠心肌细胞生长状态好,细胞存活率和自发搏动性高,操作简单,重复性好,是一种理想、可靠的原代心肌细胞培养方法。
OBJECTIVE: To establish a simple and effective method of cardiomyocyte isolation and primary culture to obtain primary cardiomyocytes with ideal quantity and activity. Methods: Under the aseptic condition, the heart of newborn SD rats were taken out within 72 hours after birth, the connective tissue and atrial tissue were removed, only the left ventricle was preserved. The myocardium was obtained by digestion with trypsin and collagenase Ⅰ and differential adherent separation The cells were cultured in vitro, the morphological changes of the cells were observed, and the statistical analysis was made on the frequency of beating and the number of beating cells. RESULTS: Isolated primary cardiomyocytes grew well and nucleoli were clearly visible. After 12 h of cardiomyocyte inoculation, a few cells adhered to the wall, and a few adherent cells appeared spontaneous beats. After that, the number of beating cells and the frequency of beating of cells increased gradually. After 48 hours of inoculation, the cells spread completely and connected Into a film, spontaneous beats appear synchronized and island-like. Conclusion: The cardiomyocytes of newborn SD rats obtained by this experiment have good growth condition, high cell survival rate and spontaneous pulsation, simple operation and good repeatability. They are an ideal and reliable primary culture method of cardiomyocytes.