BAMBI在C2C12细胞成肌分化中的作用研究

发布时间：2018-06-07 09:36

本文选题：BAMBI + Wnt/β-catenin　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：BAMBI是骨形成蛋白和激活素的跨膜抑制蛋白,其结构与转化生长因子TGF-β家族I型受体的结构相似,但胞内缺少激酶结构域。BAMBI主要通过增强Wnt/β-catenin或抑制TGF-β信号通路发挥作用,如调节细胞的增殖、迁移和分化等。Wnt/β-catenin通路又称为经典Wnt信号通路。当经典Wnt配体存在时,细胞质中的β-catenin降解复合体解体,β-catenin含量增加并转移到细胞核中,与TCF/LEF共同调控下游靶基因的表达。BAMBI能与跨膜受体Fzd5、辅助受体LRP6以及胞内蛋白Dvl2结合,增强复合物的稳定性,进而促进β-catenin的核转位和经典Wnt信号通路的转录活性。经典Wnt信号通路在胚胎肌肉发育、成年骨骼肌再生以及成肌细胞系体外增殖和分化过程中都具有重要作用。鉴于BAMBI与经典Wnt信号通路的关系,我们推测BAMBI可能调控成肌分化,但目前还没有相关报道。在本项研究中,我们首先检测了BAMBI在C2C12成肌分化过程中的时序;然后,利用小干扰RNA抑制BAMBI表达,检测成肌分化以及经典Wnt信号通路活性的变化;最后,利用Li Cl激活经典Wnt信号通路,验证BAMBI是否通过经典Wnt信号通路调控成肌分化。主要结果如下:1.BAMBI在成肌分化早期高表达。与诱导前(第0天)相比,BAMBI在诱导第1天的m RNA水平上调3倍,随后逐渐下降,直至分化结束。2.干扰BAMBI表达抑制成肌分化。与对照组相比,小干扰RNA处理组中Myo D、Myo G和My HC的表达被抑制。同时,肌管数量、分化指数和融合指数也显著下降。3.干扰BAMBI抑制经典Wnt信号通路活性。与对照组相比,小干扰RNA处理组中Axin2 m RNA水平和β-catenin核转位显著下降。4.Li Cl恢复干扰BAMBI对经典Wnt信号通路及成肌分化的抑制作用。当Li Cl存在时,干扰BAMBI不能有效降低Axin2 m RNA水平和β-catenin核转位,且干扰BAMBI所导致的成肌关键基因表达下降、肌管数量、分化指数和融合指数下降等也得到恢复。综上所述,在C2C12细胞系中,BAMBI能正调控经典Wnt信号通路和成肌分化,并且BAMBI通过增强经典Wnt信号通路促进成肌分化。
[Abstract]:BAMBI is a transmembrane inhibitor of bone morphogenetic protein and activin, and its structure is similar to that of type I receptor of TGF- 尾 family. However, the intracellular lack of kinase domain. BAMBI plays an important role by enhancing Wnt/ 尾 -catenin or inhibiting TGF- 尾 signaling pathway. The Wnt- 尾 -catenin pathway, which regulates cell proliferation, migration and differentiation, is also known as the classical Wnt signaling pathway. In the presence of classical Wnt ligands, the 尾 -catenin degradation complex in the cytoplasm disintegrates and the 尾 -catenin content is increased and transferred to the nucleus, and the expression of downstream target genes is regulated by TCF/LEF. BAMBI can bind to the transmembrane receptor Fzd5, the coreceptor LRP6 and the intracellular protein Dvl2. The stability of the complex was enhanced, which promoted the nuclear translocation of 尾 -catenin and the transcriptional activity of classical Wnt signaling pathway. Classical Wnt signaling pathway plays an important role in embryonic muscle development, adult skeletal muscle regeneration and proliferation and differentiation of myoblast in vitro. In view of the relationship between BAMBI and classical Wnt signaling pathway, we speculate that BAMBI may regulate myogenic differentiation, but there are no reports yet. In this study, we first examined the timing of BAMBI during myogenesis of C2C12; then, we used small interfering RNA to inhibit the expression of BAMBI, to detect the changes of myogenic differentiation and the activity of classical Wnt signaling pathway. The classical Wnt signaling pathway was activated by LiCl to verify whether BAMBI regulated myogenic differentiation through the classical Wnt signaling pathway. The main results were as follows: 1. High expression of BAMBI in early myogenic differentiation. Compared with pre-induction (day 0), the level of m RNA increased by 3 times on the first day of induction, and then decreased gradually until the differentiation ended. 2. Interfering with the expression of BAMBI inhibits myogenic differentiation. Compared with the control group, the expression of Myo D, Myo G and my HC in the small interfering RNA group was inhibited. At the same time, the number of myotubes, differentiation index and fusion index also decreased significantly. Interfering with BAMBI inhibits the activity of classical Wnt signaling pathway. Compared with the control group, the level of Axin2 m RNA and the nuclear translocation of 尾 -catenin in the small interfering RNA group decreased significantly. 4. LiCl restored the inhibitory effect of interfering BAMBI on the classical Wnt signaling pathway and myogenic differentiation. In the presence of LiCl, interfering BAMBI could not effectively decrease the level of Axin2 m RNA and 尾 -catenin nuclear translocation, and the decrease of myogenic key gene expression induced by interference with BAMBI could also restore the number of myotubes, differentiation index and fusion index. In conclusion, in C2C12 cell line, C2C12 can regulate the classical Wnt signaling pathway and myogenic differentiation, and BAMBI promotes myogenic differentiation by enhancing classical Wnt signaling pathway.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.2

【参考文献】