摘要
目的:改进原代乳小鼠心肌细胞的分离及培养方法,稳定获得高活性以及高纯度的原代乳小鼠心肌细胞。方法:取1~3日龄新生乳小鼠心脏,采用胰蛋白酶联合Ⅱ型胶原酶两步消化法,通过差速贴壁分离纯化心肌细胞,体外培养于含10%马血清的改良DMEM。在倒置相差显微镜下观察心肌细胞形态学特征及搏动规律,台盼蓝染色检测心肌细胞存活率,利用肌钙蛋白T(cTnT)免疫荧光染色鉴定心肌细胞。结果:原代乳小鼠心肌细胞分离培养24 h后基本贴壁,并出现自发性搏动,第4天聚合成簇,呈同步性搏动;细胞存活率平均为90.63%,心肌细胞纯度平均为93.17%。结论:本实验采用改良分离及培养方法能够稳定获得高活性以及高纯度的原代乳小鼠心肌细胞。
Objective:To improve the isolation and culture methods of primary cardiomyocytes in neonatal mouse to stably get high activity and purity of primary mouse cardiomyocytes.Methods:Hearts from 1-3-day-old neonatal mouse were harvested,and a two-step digestion method combining trypsin and typeⅡcollagenase was used.Differential adherence was then employed to purify the cardiomyocytes,which were cultured in vitro in modified Dulbecco′s Modified Eagle Medium(DMEM)containing 10%horse serum.The morphological characteristics and beating patterns of the cardiomyocytes were observed under an inverted phase-contrast microscope.Cell viability was assessed by using trypan blue staining,and cardiomyocytes were identified by immunofluorescence staining for cardiac troponin T(cTnT).Results:After 24 hours of culture,the primary neonatal mouse cardiomyocytes adhered to the culture dish and exhibited spontaneous beating.By 96 hours,the cells aggregated into clusters and displayed synchronous beating.The cell survival rate was 90.63%,and the purity of cardiomyocytes reached 93.17%.Conclusion:The modified isolation and culture methods used in this study obtained the primary neonatal mouse cardiomyocytes with high activity and purity.
作者
罗洁
曹梦菲
付可威
吕书梅
袁伟
LUO Jie;CAO Mengfei;FU Kewei;LYU Shumei;YUAN Wei(Department of Cardiology,Affiliated Hospital of Jiangsu University,Zhenjiang Jiangsu 212001,China)
出处
《江苏大学学报(医学版)》
2025年第2期161-165,共5页
Journal of Jiangsu University:Medicine Edition
基金
江苏省重点研发计划项目(BE2021694)。
关键词
新生小鼠
心肌细胞
两步酶消化法
原代培养
neonatal mouse
cardiomyocyte
two-step enzymatic digestion method
primary culture
