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目的对比研究三种乳鼠原代心肌细胞消化分离方法,比较不同方法分离获取的心肌细胞在数量、纯度、活力上的差异,为相关实验研究提供选择。方法按照不同的消化方法进行分组,A组为0.08%胰蛋白酶单纯消化组;B组为0.08%胰蛋白酶+0.08%Ⅱ型胶原酶混合消化组;C组为0.08%胰蛋白酶和0.08%Ⅱ型胶原酶分离消化组。记录差速贴壁后的细胞数和活细胞率,对贴壁24h后的心肌细胞进行免疫荧光染色,计算心肌细胞纯度,通过激光共聚焦显微镜对JC-1染色的心肌细胞进行线粒体膜电位检测,比较红绿荧光密度的差异,通过能量代谢间接反映细胞活力。结果三种方法分离获得的心肌细胞数差异无统计学意义(P>0.05),而活细胞率C组高于A组(P<0.01);三组分离获得的心肌细胞纯度差异无统计学意义(P>0.05);线粒体膜电位,A组荧光比值为0.928±0.078,B组荧光比值为0.943±0.099,C组荧光比值为1.160±0.089,三组之间差异有统计学意义(P<0.01),C组线粒体膜电位高于A组和B组(P<0.01)。结论三种消化方法在细胞数和细胞纯度上无明显差异,0.08%胰蛋白酶和0.08%Ⅱ型胶原酶分离消化的心肌细胞在活细胞率和细胞活力方面更优,可以作为常规的乳鼠心肌细胞分离方法的选择。
Objective To comparatively study the methods of digestion and isolation of primary cardiomyocytes from three kinds of suckling mice and to compare the differences in the number, purity and viability of the cardiomyocytes obtained by different methods, so as to provide a choice for related experimental studies. Methods According to different digestion methods, Group A was 0.08% trypsin digestion alone; Group B was 0.08% trypsin + 0.08% collagenase digestion; Group C was 0.08% trypsin and 0.08% Type II Collagenase digestion group. The number of differentiated cells and the percentage of viable cells were recorded. The cardiomyocytes were stained with immunofluorescence after 24 hours. The purity of cardiomyocytes was calculated. The mitochondrial membrane potential of JC-1-stained cardiomyocytes was detected by laser scanning confocal microscopy , Comparing the difference between red and green fluorescent densities, indirectly reflecting cell viability through energy metabolism. Results There was no significant difference in the number of cardiomyocytes isolated from the three methods (P> 0.05), while the percentage of viable cells in group C was higher than that in group A (P <0.01). There was no significant difference in the purity of cardiomyocytes (P> 0.05). The mitochondrial membrane potential, the fluorescent ratio of group A was 0.928 ± 0.078, the fluorescence ratio of group B was 0.943 ± 0.099, the fluorescence ratio of group C was 1.160 ± 0.089, the difference was statistically significant ). The mitochondrial membrane potential of group C was higher than that of group A and group B (P <0.01). Conclusion The three digestion methods showed no significant difference in cell number and cell purity. Cardiomyocytes isolated and digested with 0.08% trypsin and 0.08% type Ⅱ collagenase were better in terms of viable cell ratio and cell viability and could be used as routine neonatal rat cardiomyocytes Selection of Cell Separation Methods.