改良的乳小鼠心肌细胞原代培养方法

摘要

目的：建立一种稳定而快速的乳小鼠心肌细胞的原代培养方法。方法用多聚赖氨酸预包被处理培养皿，采用两步法（0.25％胰酶4℃过夜，0.5 mg／mL－1.0 mg／mL II型胶原酶＋5 mg／mL白蛋白的胶原消化液37℃短时多次消化），通过差速贴壁70 min ＋5-溴脱氧尿嘧啶的使用来纯化心肌细胞。倒置相差显微镜下观察心肌细胞形态变化，台盼蓝染色法检测细胞存活率，α-actinin特异性免疫荧光染色鉴定心肌细胞并计算纯度。结果心肌细胞形态良好，自发搏动，细胞成活率可达到98％，纯度可达到95％。结论本方法培养的心肌细胞存活率高，纯度高，是一种理想的心肌细胞原代培养方法。%Objective To establish a stable and fast method for primary culture of mouse cardiomyocytes. Methods Dishes were coated with polylysine firstly.A two-step approach was used to isolate and digest mouse cardiomyocytes cells (0.25%trypsin in 4°C overnight and 0.5 mg/mL to 1.0 mg/mL collagenase +5 mg/mL albumin collagen digestion liquid in 37°C for short-time digestion), then the cardiomyocytes were purified through differential adhesion for 70 min and 5-bromodeoxyuridine ( BrdU) .The cell morphology was observed under an inverted microscope. The survival rate of cardiacmyocytes was detected by trypan-blue staining and their purity was identified by α-actinin immunofluorescence staining.Results The cardiomyocytes were in good shape and pulsed spontaneously.The survival rate of the cardiomyocytes reached 98%and the purity was 95%.Conclusions This method described in this study is an ideal method for primary culture of mouse cardiomyocytes with a high survival rate and high purity.