改良法分离培养SD新生乳鼠原代心肌细胞  

An improved method for isolation and culture of primary cardiomyocytes from SD neonatal rats

作　　者：孟香红[1] 曾斌[1] 陈慧玲[1] 李伟东[1] 李娜 徐小勇 MENG Xiang-hong;ZENG Bin;CHEN Hui-ling;LI Wei-dong;LI Na;XU Xiao-yong(Department of Medical Technology,Ningbo College of Health Sciences,Ningbo 315104;Department of Cardiovascular Disease,Ningbo Medical Treatment Centre Li Huili Hospital,Ningbo 315040,China)

机构地区：[1]宁波卫生职业技术学院医学技术学院,浙江宁波315104 [2]宁波市医疗中心李惠利医院心血管科,浙江宁波315040

出　　处：《中国应用生理学杂志》2021年第6期699-704,共6页Chinese Journal of Applied Physiology

基　　金：浙江省教育厅科研项目(Y201738580);宁波卫生职业技术学院校级重点课题(2019Z05);宁波市自然科学基金项目(2017A610200)。

摘　　要：目的:建立一种稳定、快速的SD新生乳鼠原代心肌细胞分离培养改良方法。方法:取SD新生乳鼠心室,0.12%Ⅱ型胶原酶消化,Percoll密度梯度离心结合5-溴脱氧尿嘧啶(5-BrdU)化学抑制法纯化心肌细胞,体外培养于含5%马血清的改良DMEM/F12中,次日更换为普通含10%胎牛血清的高糖DMEM继续培养,并比较此改良方法与传统差速贴壁法的差异。结果:改良法获得的心肌细胞生长良好,接种24 h后几乎全部贴壁生长,细胞呈三角形、梭形或不规则形,个别细胞出现自主搏动,频率为10~30 beats/min不等。48 h后心肌细胞变长伸出伪足,部分细胞呈现同步搏动,频率接近50~80 beats/min。72 h后心肌细胞成菊花样交织成网,自发搏动趋于同步,频率加快至80~100 beats/min;96 h后细胞聚集成簇,呈岛屿样,同步搏动频率在100~120 beats/min左右,一周内细胞状态良好。改良法纯化原代心肌细胞的得率((1.17±0.15)×10^(6) vs(1.21±0.22)×10^(6),P>0.05)和存活率与传统差速贴壁法相当(93.3%±1.4%vs 92.2%±0.7%,P>0.05),但是改良方法获得的原代心肌细胞纯度更高(94.7%±2.1%vs 89.5%±1.3%,P<0.05),且用时较短((3.1±0.4)h vs(4.3±0.3)h,P<0.01)。结论:改良法获得心肌细胞耗时短、纯度高、结构功能保存完整,且实验重复和稳定性好,是一种理想且简单易行的的原代心肌细胞分离培养方法。Objective:To establish a stable,rapid and improved method for isolation and culture of primary cardiomyocytes from neonatal rats.Methods:Ventricular tissues from neonatal SD rats were digested with 0.12%collagenaseⅡ,and then subjected to Percoll density gradient centrifugation.The original cardiomyocytes were cultured in modified DMEM/F12 containing 5%horse serum and 5-bromodeoxyuracil(5-BrdU)in vitro for further purification,and medium was changed to normal high glucose DMEM with 10%FBS the next day.The difference between the improved method and traditional differential attachment one used for isolation and culture of primary cardiomyocytes was compared.Results:Cardiacmyocytes obtained through the improved method grew well.24 hours after plating,most cells adhered to the dishes,with shapes looked triangular,fusiform or irregular,and some of them showed spontaneously contract at a frequency varying from 10~30 times/min.After 48 h culture,the cardiomyocytes became longer and stretched out pseudopodia.Some cells showed synchronous beats with the frequency close to 50~80 times/min.72 hours later,cardiomyocytes were interwoven into a network in chrysanthemum patterns,and spontaneous beats tended to be more synchronous,with a frequency of 80-100 times/min.After 96 h,cells gathered into clusters as islands,with synchronous beat at a frequency of around 100~120 times/min.All cardiomyocytes were in good condition within one week.Yields((1.17±0.15)×106 vs(1.21±0.22)×106,P>0.05)and survival rate of primary cardiomyocytes obtained by the improved method was comparable to that gained using traditional differential attachment way(93.3%±1.4%vs 92.2%±0.7%,P>0.05),but the purity of primary cardiomyocytes obtained through the improved method was much higher(94.7%±2.1%vs 89.5%±1.3%,P<0.05),while with less time consuming((3.1±0.4)h vs(4.3±0.3)h,P<0.01).Conclusion:This improved method is an ideal and simple method for the isolation and culture of primary cardiomyocytes with shorter time-consuming,high purity,intact struc

关 键 词：新生乳鼠 原代心肌细胞 Percoll密度梯度离心 化学抑制 

分 类 号：R73-3[医药卫生—肿瘤]