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目的研究多次差速贴壁并联合应用5-溴脱氧尿苷(5-B rdU)对分离的心肌细胞纯度及活性的影响。方法以酶消化法分离乳鼠心肌细胞,分别进行1~3次差速贴壁并联用或不联用5-B rdU以纯化心肌细胞。正常培养3 d后,观察细胞的形态特征。用流式细胞技术分析各实验组心肌细胞的纯度。以台盼蓝染色、MTT比色法及细胞线粒体内膜功能检测(流式细胞术)来反映各实验组心肌细胞的活性。结果随着差速贴壁次数的增加,心肌细胞得以纯化,但细胞的活性有所下降。联用5-B rdU后细胞纯度进一步提高,且对细胞的活性无明显影响;其中两次差速贴壁并联合应用5-B rdU后,可获得纯度高、活性好的心肌细胞。结论两次差速贴壁并联合应用5-B rdU为纯化心肌细胞的有效方法。
Objective To study the effect of 5-Brodeoxyuridine (5-B rdU) on the purity and activity of isolated cardiomyocytes after multiple differential adherent and combined application. Methods Cardiomyocytes were isolated by enzymatic digestion. Cardiomyocytes were purified with or without 5-BrdU 1 ~ 3 times with differential adherent method. After 3 days of normal culture, the morphological characteristics of the cells were observed. The purity of cardiomyocytes in each experimental group was analyzed by flow cytometry. Trypan blue staining, MTT colorimetric assay and mitochondrial inner membrane function (flow cytometry) to reflect the activity of myocardial cells in each experimental group. Results With the increase of the number of differential adherent cells, cardiomyocytes were purified, but the activity of cells decreased. The cell purity was further increased after combined with 5-BrdU, and had no significant effect on the cell activity. After the two adherent cells were combined with 5-BrdU, the cells with high purity and activity were obtained. Conclusion Two differential adherent and combined 5-B rdU for the purification of myocardial cells is an effective method.