三种不同乳鼠原代心肌细胞消化分离方法的对比研究  

Three Different Digestion Methods to Separate Neonatal Rat Cardiomyocytes

作　　者：段鹏[1] 王金鑫[1] 刘晓静[1] 卫世强 段玉慧 朱庆磊[1] 

机构地区：[1]解放军总医院心内科,北京100853 [2]解放军371中心医院心内科,新乡453000

出　　处：《四川大学学报（医学版）》2017年第2期286-289,共4页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(No.81570349)资助

摘　　要：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 investigate the efficacy of three different digestion methods for the separation of neonatal rat cardiomyocytes. Methods 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 Ⅱ collagenase mixed digestion, group C was 0.08% trypsin and 0.08% type Ⅱ 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. Results There was nostatistically significant difference in the number of cells between three groups （P 〉0.05）.The rate of cell viability in group C was significantly higher than that in group A （P 〈0.01）. No statistically significant difference was found in the purity of cardiomyocytes between three groups （P 〉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 （P 〈0.01）. Conclusion The cell isolation method with 0.08% trypsin and 0.08% type Ⅱ collagenase isolated digestion could be served as conventional method to separate neonatal rat cardiomyocytes.

关 键 词：乳鼠 胰蛋白酶 Ⅱ型胶原酶 激光共聚焦显微镜 线粒体膜电位 

分 类 号：R-33[医药卫生]