新生大鼠心脏成纤维细胞体外分裂方式

Division types of cultured cardiac fibroblasts in neonatal rat

目的 观察体外培养状态下心脏成纤维细胞（CFs）的形态特征和分裂增殖方式。方法原代培养新生大鼠CFs，免疫荧光双标法分别检测波形蛋白和磷酸化组蛋白H3（H3P）共表达，波形蛋白和微管蛋白共表达。利用活细胞工作站和荧光显微镜观目的 观察处于不同分裂期的细胞时相。结果CFs 0．5～1h贴壁，培养2～3d细胞增殖旺盛，大量细胞进入有丝分裂期，其中多数CFs先隆起变圆，而少数则仍维持扁平状进行分裂。偶见无丝分裂现象。圆隆型有丝分裂时长为20～30min，扁平型分裂时长为40～50min。分裂前期胞核形状规则，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，H3P高表达，纺锤体呈典型纺锤样；后期染色单体分开并移向两极，H3P在染色单体上持续表达；末期两个子核形成，微管聚集在两子核之间；胞质分裂期，两个子细胞形成，H3P不表达。结论体外培养的CFs存在圆隆型和扁平型两种有丝分裂形态，在分裂方式上存在有丝分裂和无丝分裂两种形式。

Objective To observe the morphological features and the division types of rat cardiac fibroblasts（CFs） in vitro. Methods Primary neonatal rat CFs were cultured and double labeled by vimentin and phosphorylation of histone 3 （H3P） or tubulin. Cell phases in division stages were observed with the living cell station and fluorescence microscopy. Results CFs began to attach to the bottom of the culture flask at 0.5-1hour. After being cultured for 2-3days, the cells presented a strong proliferation ability, as lots of elevated round cells entered mitosis stage, and a few CFs remained fiat in division stage. At the same time, there were occasionally amitosis phenomenons in CFs. The splitting time of the elevated mitosis lasted for about 20-30min, while the fiat type 40-50min. In the prophase, the cell nucleus had a regular shape and H3P began to express; in the prometaphase, the nuclear envelope broke and the spindle formed; in the metaphase, chromosomes aligned on the equatorial plane, and H3P had a high expression in the chromosome, and the spindle showed its typical form; in the telophase, the chromotid divided and moved towards the two poles, and H3P split with it; then the two daughtere＇ nuclei formed with microtubule coalescence between them; finally, the cytoplasm split into two daughter ceils and H3P disappeared. Conclusion There are two division morphologies-elevated and flat, and two division types- mitosis and amitosis,of CFs cultured in vitro.