流式细胞术测定骨骼肌细胞分化中DNA合成及其细胞周期变化

Analysis of cell cycle and DNA synthesis during myocytic differentiation by flowcytometry

目的 分析骨骼肌细胞分化中细胞周期及其对 DNA合成的影响 .方法 采用小鼠 C2 C12骨骼肌细胞系作为体外肌细胞分化的研究模型 ,对照组 10 0 m L· L- 1胎牛血清DMEM培养液培养 ,实验组换为 2 0 m L· L- 1 胎牛血清DMEM培养液培养 ,诱导肌母细胞分化 ,分别于换 2 0 m L·L- 1 血清浓度 DMEM培养液后的 2 4,48和 72 h进行流式细胞术检测 ,分析肌母细胞分化过程中 DNA合成和细胞周期变化 .结果 发现肌细胞分化早期即 2 4,48h时 ,与对照组比有更多的细胞进入 S期 ;分化后期即 72 h多核肌管形成时 ,S期比对照组明显减少 ,而 G0 / G1 期明显高出对照 .此外 ,在肌分化的 72 h内 G0 / G1 期细胞随时间而递增 ,S期则递减 .结论 上述现象说明随着细胞向成熟分化的进程 ,肌细胞融合成肌管 ,处于 DNA合成复制期的细胞逐渐减小 ,更多的细胞进入相对稳定的 G0 / G1 期 .可见肌细胞的生成和分化是一个不可逆的、高度有序的过程 .

AIM To investigate cell cycle and DNA synthesisduring myocytic differentiation. METHODS Murine C2C12 myoblasts were used in this experiment. The control group was cultured in Dulbeccco's modified eagle's medium (DMEM) supplemented with 100 mL·L -1 fetal bovine serum (FBS). Proliferating myoblasts were shifted to DMEM supplemented with 20 mL·L -1 FBS for 24, 48 or 72 hours to induce differentiation, and were then analyzed by flow cytometry. RESULTS In contrast with the control, more cells of the experimental group entered S phase during the early differentiation (24 h, 48 h), whereas a smaller number of cells entered (what ) during the late differentiation (72 h) when multinucleated myotubes began to form. Besides, G 0 / G 1 phase cells increased with the time, whereas S phase cells decreased during differentiation. CONCLUSION These data demonstrate that during the differentiation process, myoblasts fuse to form myotubes; the cells decrease at the phase of DNA duplication, and a greater number of cells enter a relatively steady G 0 / G 1 phase. Therefore, skeletal myogenesis and differentiation can be considered as a highly ordered and irreversible process.