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体外培养状态下心肌细胞有丝分裂的证据
Evidence of rat cardiomyocyte mitosis in vitro
目的 探讨体外培养状态下心肌细胞有丝分裂、增殖的证据。 方法 分离新生大鼠心肌细胞，分别原代培养0h至12d；免疫荧光分别双标心肌肌钙蛋白-T（cTnT）、磷酸化组蛋白H3（H3P）和微管蛋白（tubulin），利用活细胞工作站和荧光显微镜观察处于分裂期的心肌细胞。 结果 原代培养的心肌细胞生长状态好、纯度高，可观察到分裂各期的心肌细胞及其微管蛋白的变化。处于分裂各期的心肌细胞不隆起变圆仍维持扁平状。分裂前期细胞核形状规则，染色质凝集，H3P开始表达；前中期核膜破裂，纺锤体形成；中期染色体排列在赤道面，纺锤体呈典型纺锤样；后期染色单体分开并移向两极；末期子核形成，平行微管聚结在两子核之间；胞质分裂期形成两个子细胞，两个细胞分开后，可见有少量微管呈丝状连接。原代培养1～12d的心肌细胞在整个细胞分裂图像中很少见到双核心肌细胞。培养第3天开始发现分裂象的心肌细胞,占总心肌细胞数的2/127(1.57%)和1/92(1.09%)，培养第7天，分裂细胞比率达3/69(4.35%)和5/163(3.07%)，培养第12天有2/100(2%)和0/60(0)的心肌细胞处于分裂期。 结论 体外培养的心肌细胞存在一定数量的完全有丝分裂，是心肌增殖和心肌再生的细胞学证据。
Objectiv To inverstigate myocardial cell mitosis and proliferation in vitro. Methods Primary neonatal rat cardiomyocytes were cultured from 0 hour to 12 days and 5 days, and double stained by cardiac tropnin-T (cTnT), phospho histone H3 (H3P) and tubulin using immunofluorescence technique. Cardiomyocytes with mitosis were searched with the living cell station and a fluorescence microscope. Results Primary cardiomyocytes were lived well and had a high purity. The tubulin change at every mitosis period was observed. The cardiomyocytes were not swelled up and maintained squamous under mitosis condition. The nuclear shape in mitosis prophase was regular, the chromatin became condensation and H3P began to express; At prometaphase, karyotheca broken and spindle formed; Chromosomes were arranged in the equatorial plane and the shape of spindle was very typical at metaphase. The character at anaphase was that the chromatids separated and moved towards the poles and the spindle extended. At telophase, daughter nucleus came into being and parallel microtubule distributed between two daughter nucleus; At cytokinesis period, two daughter cells were generated and a small amount of silky microtubules located between two daughter cells were seen after the two daughter cells separation. The conjugate nuclei of primary cultured neonatal rat cardiomyocytes from 1 day to 12 days were seldom observed in all cell division image. The division phase of cardiomyocytes was observed and the proportions of division cell were approximately 2/127(1.57%) and 1/92(1.09%) at cultured 3 days, 3/69(4.35%) and 5/163(3.07%) at 7 days, 2/100(2%) and 0/60(0) at 12 days. Conclusion Cardiomyocyte in vitro exists completing mitosis division in few cells which is cell evidence of cardiac regeneration and proliferation.
有丝分裂 / 心肌细胞 / 免疫荧光技术 / 体外培养 / 大鼠
Mitosis / Cardiomyocyte / Immunofluorescence / Cell culture / Rat
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