Duan Peng, Wang Jin-Xin, Liu Xiao-Jing, Wei Shi-Qiang, Duan Yu-Hui, Zhu Qing-Lei
Department of Cardiovascular, Chinese PLA General Hospital, Beijing 100853, China;2.
371 Hospital, Xinxiang 453000, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2017 Mar;48(2):286-289.
To investigate the efficacy of three different digestion methods for the separation of neonatal rat cardiomyocytes.
We employed three different digestion methods to separate neonatal rat cardiomyocytes. Group A was 0.08% trypsin digestion alone, group B was 0.08% trypsin+0.08% type 2 collagenase mixed digestion, group C was 0.08% trypsin and 0.08% type 2 collagenase isolated digestion. The number of cells and cell viability after differential adhesion were recorded. The purity of cardiomyocytes was evaluated by immunofluorescence staining. The cell vitality was assessed by the detection of mitochondrial membrane potential with JC-1 staining, and the ratio of red to green fluorescence intensity by laser scanning confocal microscope.
statistically significant difference in the number of cells between three groups ( >0.05).The rate of cell viability in group C was significantly higher than that in group A ( <0.01). No statistically significant difference was found in the purity of cardiomyocytes between three groups ( >0.05). The ratio of red to green fluorescence intensity in group A, B and C were 0.928±0.078, 0.943±0.099 and 1.160±0.089, respectively; the ratio in group C was significantly higher than that in group A and group B ( <0.01).
The cell isolation method with 0.08% trypsin and 0.08% type 2 collagenase isolated digestion could be served as conventional method to separate neonatal rat cardiomyocytes.
探讨三种不同消化方法分离新生大鼠心肌细胞的效果。
采用三种不同消化方法分离新生大鼠心肌细胞。A组为单纯0.08%胰蛋白酶消化，B组为0.08%胰蛋白酶+0.08%Ⅱ型胶原酶混合消化，C组为0.08%胰蛋白酶和0.08%Ⅱ型胶原酶分步消化。记录差速贴壁后细胞数量及细胞活力。通过免疫荧光染色评估心肌细胞纯度。采用JC-1染色检测线粒体膜电位评估细胞活力，并通过激光扫描共聚焦显微镜检测红绿荧光强度比值。
三组细胞数量差异无统计学意义（>0.05）。C组细胞活力率显著高于A组（<0.01）。三组心肌细胞纯度差异无统计学意义（>0.05）。A、B、C组红绿荧光强度比值分别为0.928±0.078、0.943±0.099和1.160±0.089；C组比值显著高于A组和B组（<0.01）。
0.08%胰蛋白酶和0.08%Ⅱ型胶原酶分步消化的细胞分离方法可作为分离新生大鼠心肌细胞的常规方法。
