新生SD大鼠心肌细胞分离培养及纯化的快速便捷方法及鉴定.docVIP

新生SD大鼠心肌细胞分离培养及纯化的快速便捷方法及鉴定 付微 刘飞 乔陆明 张绪东 牡丹江医学院 牡丹江市第二人民医院 X 关注成功！ 加关注后您将方便地在 我的关注中得到本文献的被引频次变化的通知！ 新浪微博 腾讯微博 人人网 开心网 豆瓣网 网易微博 摘????要： 目的 探讨一种体外培养存活率高、纯度高、耗时少的心肌细胞改良方法。方法 采用自配0.25%胰蛋白酶重复消化新生24h以内乳鼠的心室组织多次, 除第一次收集的上清外, 其余收集到的细胞用含12%胎牛血清的高糖DMEM培养基中和以终止消化, 差速贴壁90min, 加入终浓度为0.1mmol/L的5-溴-2-脱氧尿苷 (5-Brdu) 纯化心肌细胞。结果 联合应用差速贴壁分离法和抑制成纤维细胞增殖的方法培养出的心肌细胞于倒置显微镜下观察其细胞生长状态良好, 24h后基本全部贴壁, 心肌细胞呈梭形、三角形, 不规则形等, 搏动频率在50100次/min, 持续时间久, 活力好且稳定。经心肌肌钙蛋白免疫荧光鉴定心肌细胞纯度为92.9%。结论 本研究应用改良的新生乳鼠心肌细胞培养方法, 快速便捷, 得到存活率较高、纯度高、搏动状态良好的心肌细胞, 且培养耗时短, 可重复性高。可为研究心脏疾病的基础科学研究提供理想的实验模型。 关键词： 心肌细胞; 免疫荧光; 原代培养; 方法改良; 快速便捷; 作者简介：付微 (1991-) , 女, 汉, 在读硕士, 主要从事生理学研究, E-mail:943498398@。 作者简介：张绪东, 教授, 硕士生导师, E-mail。 收稿日期：2017-08-19 基金：黑龙江省创新训练项目 (201510229014) Improvement of Primary Cardiomyocyte Culture in Newborn SD Rats FU Wei School of Basic Medical Sciences Mudanjiang Medical University; Abstract： Objective To explore a method for improving cardiomyocytes with high survival rate, high purity and low time in vitro culture.Methods Using self-contained 0.25% trypsin repeated digestion 24 h 24 h neonatal ventricular tissue several times, The remaining collected cells were neutralized with high glucose DMEM medium containing 12% fetal bovine serum, except for the first supernatant collected Termination of digestion, differential adherent 90 min, adding the final concentration of 0.1 mmol/L 5-bromo-2-deoxyuridine (5-Brdu) purification of cardiomyocytes.Results The cardiomyocytes cultured under the method of differential adherence separation and inhibition of fibroblast proliferation were observed under inverted microscope.The cells were well adhered under24 h, and the myocardium was spindle-shaped, triangular, irregular And so on, the pulse frequency of 50-100 times/min, lasting a long time, good vitality and stability.The purity of myocardial cells was 92.9% by cardiac troponin immunofluorescence.Conclusion In this study, the modified neonatal rat cardiomyocyte culture method was used to obt