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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(c Tn T)、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。
Objective To investigate the evidence of mitosis and proliferation of cardiomyocytes cultured in vitro. Methods Cardiac myocytes from neonatal rats were isolated and cultured for 0h to 12d respectively. The double labeled cardiac troponin T (cTnT), phosphorylated histone H3 (H3P) and tubulin were detected by immunofluorescence. Live cell workstation and fluorescence microscopy in the mitotic cardiomyocytes. Results Primary cultured cardiomyocytes grew well and had high purity. Changes of myocardial cells and their tubulins at each stage of division were observed. The cardiomyocytes in the period of division did not bulge and remained flat. The nucleus of pre-mitosis had regular shape, chromatin condensation, H3P began to express; the rupture of nuclear membrane in the middle and early stages, spindle formation; metaphase chromosome arranged in the equatorial plane, the spindle was typical spindle-like; late chromophore separated and moved to the bipolar; Nuclear formation, parallel microtubules coalescence between the two nuclear; cytokinesis formation of two daughter cells, the two cells separated, we can see a small amount of microtubules were filamentous connection. Primary cultured cardiomyocytes from 1 to 12 days seldom see dual-core myocytes in the whole cell division image. Myocardial cells were found on the third day of culture, accounting for 2/127 (1.57%) and 1/92 (1.09%) of the total number of myocytes. On day 7, the rate of dividing cells reached 3/69 (4.35%), And 5/163 (3.07%). On day 12, 2/100 (2%) and 0/60 (0) of cardiomyocytes were dividing. Conclusion There is a certain number of complete mitosis in cultured cardiomyocytes, which is cytological evidence of myocardial proliferation and myocardial regeneration.