改良的乳鼠心肌细胞分离与培养新方法.docxVIP

广东药学院学报Journal 广东药学院学报 Journal of Guangdong Pharmaceutical University Jun．2015．31(3) 改良的乳鼠心肌细胞分离与培养新方法 黄礼杰，陈耀旭，杨莹莹，殷春霞，陈睿，韩福平，唐羽欣，谭文 (华南理工大学生物科学与工程学院／生物医药前孵化器／广东省发酵与酶工程重点实验室， 广东广州510006) 摘要：目的探讨一种简单有效的sD大鼠乳鼠心肌细胞分离与培养方法。方法取出生24 h以内的大鼠 心室．O．08％的胰蛋白酶反复消化多次，离心收集心肌细胞，差速培养后5一溴脱氧尿嘧啶核苷纯化心肌 细胞。用全细胞膜片钳方法，记录心肌细胞动作电位，并用激光共聚焦显微镜观察钙瞬变、线粒体膜 电位。结果心肌细胞存活率大于93％，心肌细胞纯度大于96％，细胞形态良好，出现节律性搏动，钙瞬 变波形稳定、规律，动作电位和线粒体膜电位均正常。结论本研究方法分离心肌细胞存活率高、活性 好，是一种简单、高效的心肌细胞原代培养方法，可广泛应用于各种研究。 关键词：新生大鼠；心肌细胞；原代培养；钙瞬变；线粒体膜电位；动作电位 中图分类号：R一331文献标志码：A doi：10．3969／j．issn．1006—8783．2015．03．023 文章编号：1006—8783(2015)03—0388—05 An improved method for isolation and culture of neonatal rat cardiomyocytes HUANGⅡjie，CHEN Yaoxu，YANG Yingying，YIN ChurLxia，CHEN Rui，HAN Fuping，TANG Yuxin，TAN Wen (School ofBioscience and Bioengineering，South China University of Technology，Guangzhou 5 10006，China； Pre—incubator for Innovative Drugs and Medicine Center，South China University of Technology，Guangzhou 510006，China；Guangdong Provincial Key Laboratory ofFermentation and Enzyme Engineering，Guangzhou 510006，China) Abstract：Objective To explore simple and effective primary culture method of neonatal rat cardiomyocytes．Methods The ventricular myocardium was removed from neonatal within 24 h after birth．The cardiomyocytes were separated by repeated digestion with 0．08％trypsin and collected by centrifugation．Differential adhesion method and 5-bromodeoxyuridine(BrdU)were used in purifying cardiomyocytes．The action potential was measured by whole cell patch—clamp．The calcium transients and mitochondrial membrane potential of cardiomyocytes were observed by confocal microscope．Results The cell viability was more than 93％．The purity of cardiomyocytes was more than 96％．The morphology of cardiomyocytes appeared normal．The calcium transients were steady and regular．The mitochondrial membrane potential and action potential of cardiomyocytes were normal．Conclu