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/m L-1.0 mg/m L II型胶原酶+5 mg/m L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 / m L to 1. 0 mg / m L collagenase ＋ 5 mg / m L 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（ Brd U）. 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.
出处
《中国比较医学杂志》
CAS
北大核心
2016年第4期62-67,共6页
Chinese Journal of Comparative Medicine
基金
国家自然科学基金(编号:31300946
31260250)
沪州市人民政府-沪州医学院科技战略合作科技项目(2013LZLY-J22)
关键词
乳小鼠
心肌细胞
原代培养
Neonatal mouse
Cardiomyocytes
Primary cell culture
