【摘要】 目的观察生后不同时间乳鼠心肌细胞的形态结构和功能特征,建立理想的分离和纯化乳鼠心肌细胞技术。方法采用胰蛋白酶消化和机械分离,结合两次差速贴壁法以及使用溴脱氧尿嘧啶核苷(BrdU),从生后1~7d乳鼠心脏分离和纯化心肌细胞。观察细胞的形态和自发性搏动,并用心肌特异性肌钙蛋白丁(cTnT)免疫细胞化学染色进行鉴定。利用原位透射电镜术观察细胞超微结构,并观察其对肾上腺素和异丙肾上腺素的反应。结果从乳鼠心脏分离的细胞95%以上为心肌细胞,存活率为95%以上。乳鼠心肌细胞包括短柱状或杆状细胞和不规则形细胞。生后1~3d鼠大部分杆状细胞呈现幼稚心肌细胞的超微结构特征。6~7d鼠的杆状细胞具有类似于成熟心肌细胞的超微结构特征,cTnT呈高表达,横纹明显。不规则形细胞含有少量肌丝束,排列不规则。少数体积较小的细胞呈现未分化细胞的超微结构特征。培养72h后80%以上的细胞出现自发性搏动。肾上腺素和异丙肾上腺素刺激后,搏动细胞数目增多,搏动增强。生后6~7d鼠杆状细胞的药物反应较强。结论两类心肌细胞的超微结构特征、自发性搏动和对肾上腺素和异丙肾上腺素的反应不同。生后6~7d鼠心肌细胞较适合于成熟心肌细胞的体外实验研究。本实验建立的方法是一种较理想的乳鼠心肌细胞培养方法。更多还原

【Abstract】 Objective To investigate morphologic,structural and functional characteristics of cardiomyocytes from neonate rats and to set up a desirable technique for isolating and purifying the cardiomyocytes from neonate rats. Methods Using trypsin-digestion,mechanical separation,twice seedings and bromodeoxyuridine(BrdU)-treatment,the cardiomyocytes were isolated and purified from the hearts of neonate rats at 1-7 days after birth.Shapes and spontaneous pulsation of the cells were viewed.The cells were identified with cardiac isoform of tropnin T(cTnT) immunostaining.Ultrastructural features of the cells were examined with in situ transmission electron microscopy.Responses of the cells to adenine and isoprenaline were also examined. Results More than 95% cells isolated from the hearts of neonate rats are cardiomyocytes.The vital cells are more than 95%.Neonate rat cardiomyocytes include short columnar or rhabdoid cells and irregular cells.The most rhabdoid cells from the rats at 1-3 days after birth present the ultrastructural features of immature cardiomyocytes.The rhabdoid cells from the rats at 6-7 days after birth have some ultrastructural features of mature cardiomyocytes.Comparing with the cells at 1-3 days after birth,cTnT expression in the cells is slightly enhanced,the transverse striation was obvious.The irregular cells contain less bundles of myofilaments,the filaments are arranged irregularly.There are a few small cells which are in undifferentiated state.More than 80% cells show spontaneous pulsation at 72 hours after incubation.After treatment with adrenine and isoprenaline,the number of the cells with spontaneous pulsation increases and the intension of spontaneous pulsation is enhanced.The responses of the rhabdoid cells from the rats at 6-7 days after birth to adenine and isoprenaline are much stronger.Conclusion There are two kinds of neonate rat cardiomyocytes.They are different in ultrastructures,spontaneous pulsation and responses to adenine and isoprenaline.The cardiomyocytes from rats at 6-7 days after birth are suitable for experiments in vitro as mature cardiomyocyte.The method set up in this experiment is desirable for culture of neonate rat cardiomyocytes.更多还原