目的 观察体外培养状态下心肌成纤维细胞（CFs）的形态特征和分裂增殖方式，为研究心肌再生的机制和心肌损伤治疗提供细胞学基础和理论依据。方法 原代培养新生大鼠CFs，免疫荧光双标波形蛋白和磷酸化组蛋白H3（H3P）、波形蛋白和微管蛋白。利用活细胞工作站、扫描电镜和荧光显微镜观察处于分裂期的细胞时相。结果 CFs培养0.5～1h贴壁伸出伪足，生长状态好、形态多样，双核多，培养3～4d细胞增殖力旺盛，可见大量隆起变圆的细胞进入有丝分裂期；少数CFs分裂期仍维持扁平状；偶见无丝分裂现象。圆隆型有丝分裂时长约为20～30min；扁平型分裂时程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在染色体上高表达，纺锤体呈典型纺锤样；后期染色单体分开并移向两极，H3P随染色单体分裂；末期子核形成，微管聚结在两子核之间；胞质分裂形成两子细胞，H3P消失。结论 体外培养的CFs存在圆隆型和扁平型两种分裂形态，在分裂方式上存在有丝分裂和无丝分裂两种形式。不同的分裂形式在功能上可能存在互补。
Abstract
Objective To explore the morphological features and the division mode of rat cardiac fibroblasts cultured in vitro and to provide the cellular bases for further research of cardiac regeneration mechanism and myocardial damage therapy. Methods Primary neonatal rat CFs were cultured and double labeled by vimentin and H3P, vimentin and Tubulin respectively using immunofluorescence technique. Then mitosis CFs were searched with living cell station, scanning electron microscope and a fluorescence microscope. Results Primary cultured CFs were stick wall and extended pseudopodia within 0.5-1h, keeping a great growing state with varied shapes and were rich in binucleated cells. These cells became fecund after 3 - 4 days and it is easy to find a large number of cells which were swelled up and round into mitosis phase. A few CFs in mitosis still maintained flat,and amitosis was observed incidentally. The mitosis phase of CFs in round and swelled shape was lasted about 20-30min; while for those flat ones, it was about 40-50min.In the prophase of the mitosis, the nucleus was in regular shape, and H3P began to express; then the karyotheca broke and spindle was formed in prometaphase. At metaphase, chromosomes were found in the equatorial plane, in which H3P was expressed actively. In this phase, spindles were typically in the shape of spindle. The chromatids were separated then moved towards the cellular poles; and H3P split with their separation in the anaphase. In the telophase, two daughter nuclei were formed and between them, microtubules were distributed. At cytokinesis period, two daughter cells were generated and H3P dissolved. Conclusion CFs cultured in vitro have two different morphological features – flat, round and swelled, and two division modes- amitosis and mitosis. And it is believed that these two division modes could be complementary.
关键词
新生大鼠心脏成纤维细胞体外分裂方式
Key words
Division Mode of Cultured Rat Cardiac Fibroblast in vitro
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