关键词: 心肌细胞, 超微结构, 自发性搏动, 细胞培养, 免疫细胞化学染色, 原位透射电镜术, 乳鼠

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 EM>in situ/EM> 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 EM>in vitro/EM> as mature cardiomyocyte. The method set up in this experiment is desirable for culture of neonate rat cardiomyocytes.

Key words: Cardiomyocyte, Ultrastructure, Spontaneous pulsation, Cell culture, Immunocytochemical staining, EM>In situ/EM> transmission electron microscopy, Neonate rat