基于密度梯度法分离纯化的改良培养新生小鼠心肌细胞方法研究

Study on the modified method in culturing neonatal mouse cardiomyocytes based on density gradient method isolation and purification

目的基于Percoll不连续密度梯度法分离纯化心肌细胞,建立一个快速稳定提取新生小鼠心肌细胞的原代培养方法。方法在无菌状态下取C57BL/6乳鼠心室,用低浓度Ⅱ型胶原酶消化,通过调整温度、消化方式、离心时间及转速,采用Percoll密度梯度法分离纯化心肌细胞。观察不同时间心肌细胞形态;用0.4%台盼蓝染色检测细胞存活率及细胞数量;采用免疫荧光染色法检测心肌肌钙蛋白I(cTnI)表达情况,鉴定心肌细胞及其纯度。结果5次培养结果显示,心肌细胞的平均存活率为(93.12±0.75)%,纯度为(96.36±0.60)%,细胞生长状态良好,可以贴壁自发搏动。结论采用本方法能获得产量、存活率及纯度很高的新生小鼠原代心肌细胞,耗时较短,是一种理想的分离纯化原代心肌细胞的方法,可满足后续各种实验要求。

Objective To establish a primary culture method for rapid and stable extraction of neonatal mouse cardiomyocytes based on Percoll discontinuous density gradient method in separating and purifying cardiomyocytes.Methods The ventricle of C57BL/6 suckling mouse was taken in sterile condition and digested with low concentration collagenase II.The cardiomyocytes were separated and purified using Percoll density gradient method by adjusting the temperature,digestion method,centrifugation time and rotation speed.The morphology of cardiomyocytes at different times was observed;with 0.4%trypan blue staining,the cell survival rate and cell numbers were tested;meanwhile,immunofluorescence staining was used to detect the expression of cardiac troponin I(cTnI),and to identify cardiomyocytes and their purity.Results The five-time culture results showed that the average survival rate of cardiomyocytes was(93.12±0.75)%,and the purity was(96.36±0.60)%.The cells grew well,and could form adhesion and spontaneous beating.Conclusion The method can obtain the primary cardiomyocytes of neonatal mouse with high yield,survival rate and purity,with a short time-consuming,and thus it is an ideal method to separate and purify the primary cardiomyocytes,and can meet the requirements of subsequent.