采用Percoll非连续密度梯度离心法纯化大鼠乳鼠心室肌细胞

Enrichment of cardiomyocytes from neonatal rat ventricular tissues by discontinuous percoll gradient centrifugation

目的 探索从心室肌组织中纯化心肌细胞的方法。方法 用胰蛋白酶消化大鼠乳鼠心室肌组织 ,获得细胞悬液经Percoll分离 ,通过光镜和免疫组织化学方法对获得的每层细胞进行分析 ,采用免疫组织化学和激光共聚焦方法鉴定心肌细胞。结果 Percoll分离后 ,细胞分为 6层 ,每层细胞培养 3d时 ,通过光镜和免疫组织化学观察发现心肌细胞富集在第 4、5两层。富集的心肌细胞第 3天形成细胞簇或细胞单层 ,收缩明显而有力。心肌细胞可维持良好的形态和活力达 2～ 3个月。结论 使用Percoll分离法可以从大鼠乳鼠心室肌组织中纯化心肌细胞。

Objective To find a method for enrichment of cardiomyocytes from neonatal rat ventricular tissues.Methods Neonatal rat ventricular tissues were digested by trypsin.After discontinuous percoll gradient centrifugation,each cell layer was examined by light microscopy and immunohistochemistry.Cardiomyocytes were identified by immunohistochemistry and Confocal Laser Scanning Microscopy (CLSM).Results After discontinuous percoll gradient centrifugation,6 cell layers were formed.The majority of cardiomyocytes were identified in the fourth and fifth layers by light microscopy and immunohistochemistry.These cardiomyocytes spread over in the first day with individual cells beating spontaneously.On the day 3,the cells were arranged in loose and interlacing networks of single cells.High-power CLSM demonstrated that cell bundles mainly consisted of cardiomyocytes with sarcomeres in registry.These cells beat spontaneously and remained in good morphology and vitality 2 or 3 months later.Conclusion Cardiomyocytes disassociated from neonatal rat ventricular tissues can be enriched by method of discontinuous percoll gradient centrifugation.