【摘要】 目的 探讨过表达Bmal1对C2C12成肌细胞增殖与分化的影响及可能机制。方法 慢病毒介导成肌细胞过表达Bmal1,RT-qPCR和Western blot检测肌调节因子的表达；RNA转录组测序结合聚类分析及相关实验探究过表达Bmal1成肌细胞增殖与分化过程中Wnt信号通路和细胞周期调节因子的表达；抑制剂XAV939阻断Wnt信号通路，观察XAV939对过表达Bmal1成肌细胞增殖与分化的影响。结果 过表达Bmal1促进成肌细胞增殖，表现为S相分数、DNA增殖指数升高，EdU阳性细胞比例及β-catenin核转位率增加；诱导成肌分化，过表达Bmal1促进肌管融合，上调MyoD、myogenin、Myh3的表达。聚类分析和实验支持过表达Bmal1成肌细胞增殖与分化过程中Wnt信号传导增强，其下游细胞周期调节因子表达上调；XAV939(5μmol/L)显著下调过表达Bmal1成肌细胞Tcf1、c-Jun的表达，持续抑制过表达Bmal1成肌细胞的肌管融合，下调MyoD、myogenin、Myh3的表达。结论 Bmal1可作为骨骼肌损伤修复的靶点，过表达Bmal1通过增强Wnt信号传导促进成肌细胞增殖与分化。更多还原

【Abstract】 Objective To investigate the effect and mechanism of Bmal1 overexpression on C2C12 myoblasts proliferation and differentiation. Methods Bmal1 was overexpressed in C2C12 myoblasts by lentivirus-mediated stable transfection. The expression levels of mRNA and protein of some myogenic regulatory factors were determined by RT-qPCR or Western blot assay. Transcriptome sequence analysis and its related experiments were used to explore the expression of Wnt signaling pathway and cell cycle regulators in C2C12 myoblasts after overexpression of Bmal1 during the differentiation process. Subsequently, Wnt signaling pathway was blocked by inhibitor XAV939. The effects of XAV939 on C2C12 myoblasts after overexpression of Bmal1 were observed using corresponding experiments. Results Overexpression of Bmal1 promoted the proliferation of myoblasts cultured in growth medium. The S-phase fraction of total cells(SPF) and Proliferation Index(PI) of myoblasts increased. Compared with the blank control group and negative control group, the ratio of EdU positive cells and nuclear translocation of β-catenin were increased in C2C12 myoblasts after overexpression of Bmal1. Overexpression of Bmal1 significantly promoted myogenic differentiation and myotube fusion. The mRNA and protein expression levels of MyoD, myogenin, and Myh3were up-regulated in a time-dependent manner in C2C12 myoblasts after overexpression of Bmal1. Cluster analysis and related experiments supported that the expression of the relative factors of Wnt signaling pathway and cell cycle was up-regulated during the proliferation and differentiation of Bmal1-overexpressed C2C12 myoblasts. XAV939(5μmol/L) treatment inhibited the Bmal1 overexpression-induced the up-regulated expression of Tcf1 and c-Jun. Continuous XAV939 treatment suppressed myotube fusion and down-regulated the expression of MyoD, myogenin, and Myh3 in Bmal1-overexpressed C2C12 myoblasts. Conclusions Bmal1 may serve as a potential target for skeletal muscle injury repair.Overexpression of Bmal1 enhances myoblasts proliferation and differentiation by enhancing Wnt signaling pathway.更多还原