KLF7基因调控肌肉卫星细胞静息的分子机制研究

发布时间：2018-01-15 05:18

本文关键词：KLF7基因调控肌肉卫星细胞静息的分子机制研究　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

更多相关文章： KLF7 肌肉卫星细胞 静息 TGF-β Notch

【摘要】：骨骼肌具有强大的再生能力,肌肉损伤后需要募集前体干细胞到受伤部位,完成肌纤维的修复和生成,在成年骨骼肌中,肌肉卫星细胞行使此功能。肌肉卫星细胞是特异性肌肉干细胞,因其解剖位置位于肌纤维膜和基底膜之间而得名。在健康的成年动物中,肌肉卫星细胞维持静息状态,当受到生理或病理刺激下,能够快速激活,进行扩增及不对称分裂,完成自我更新,形成新肌纤维。维持肌肉卫星细胞静息态对于保持肌肉干细胞群数量及再生潜能意义重大。越来越多证据表明,多种胞外信号参与肌肉卫星细胞静息调节,当静息态调控因子失活或信号通路被阻断时,肌肉卫星细胞生理功能发生严重紊乱,肌肉再生能力显著下降。Krüppel家族(KLFs)多种因子参与肌肉细胞生理调控,而KLF7在神经发育及髓细胞增殖中发挥重要调节作用,表达于骨骼肌及成肌细胞,但其调控作用及机制还未探明。本研究通过体外培养小鼠肌肉卫星细胞及单根肌纤维,深入分析KLF7在肌肉细胞的调节功能。试验结果发现:(1)一周龄小鼠肌肉卫星细胞多数为激活态,而成年小鼠肌肉卫星细胞大多处于静息态,实时定量PCR分析发现KLF7在静息态肌肉卫星细胞中表达较高,蛋白表达分析证实了相似的表达趋势。在成肌细胞的研究显示,KLF7在非增殖细胞中,表达水平较高。(2)干扰KLF7促进肌肉卫星细胞激活、扩增,以及成肌细胞增殖,而过表达KLF7促进成肌细胞细胞周期阻滞,表明KLF7是调控肌肉卫星细胞静息的关键因子。(3)成肌细胞中,KLF7调控p21的表达,而干扰p21抑制KLF7对肌肉卫星细胞激活和扩增的调控作用。(4)乙酰化修饰位点突变后,KLF7对C2C12成肌细胞增殖和p21表达的调控作用受到抑制,双荧光素酶报告分析证实,乙酰化位点突变导致KLF7对p21启动子的调控显著降低,表明KLF7乙酰化位点Lys227和/或Lys231,对其在成肌细胞调控中起重要作用。(5)成肌细胞中,TGF-β信号通路负调控KLF7的表达,干扰KLF7后,TGF-β信号通路阻断诱导的肌肉卫星细胞静息和成肌细胞增殖抑制部分丧失,支持KLF7是TGF-β信号通路阻断诱导肌肉卫星细胞静息的必需因子。(6)肌肉细胞中激活经典Notch信号通路,KLF7表达上调,而干扰KLF7至少部分移除经典Notch信号通路激活诱导的肌肉细胞细胞增殖抑制,表明KLF7介导了经典Notch信号通路调节的肌肉细胞静息。(7)干扰KLF7对成肌细胞成肌分化因子和标记基因表达没有影响,暗示KLF7可能没有参与成肌分化调控。综合分析上述试验结果,得出以下结论:1)KLF7在肌肉卫星细胞静息调控中起关键作用,并抑制成肌细胞细胞周期进行。2)KLF7调控肌肉细胞激活和增殖依赖于细胞周期抑制因子p21。3)KLF7蛋白的Lys227和Lys231位点,对自身功能激活非常重要,可能存在乙酰化修饰。4)KLF7介导了胞外信号(经典TGF-β和Notch信号通路)对肌肉卫星细胞静息的调节。
[Abstract]:Skeletal muscle has a strong regeneration ability. After muscle injury, we need to recruit precursor stem cells to the injured site, complete the repair and production of muscle fiber, in adult skeletal muscle. Muscle satellite cells perform this function. Muscle satellite cells are specific muscle stem cells named for their anatomical location between the myofibroma and the basement membrane in healthy adult animals. Muscle satellite cells maintain a resting state, when stimulated by physiological or pathological, can be quickly activated, amplification and asymmetric division, complete self-renewal. It is important to maintain the resting state of muscle satellite cells in order to maintain the number and regeneration potential of muscle stem cells. There is more and more evidence that a variety of extracellular signals are involved in the resting regulation of muscle satellite cells. When the resting regulatory factor is inactivated or the signal pathway is blocked, the physiological function of muscle satellite cells is seriously disturbed. The ability of muscle regeneration decreased significantly. Kr 眉 ppel family (KLFs) involved in the physiological regulation of muscle cells, while KLF7 played an important role in neurodevelopment and myeloid cell proliferation. It is expressed in skeletal muscle and myoblast, but its regulation and mechanism have not been proved. In this study, mouse muscle satellite cells and single muscle fiber were cultured in vitro. The results showed that most of the muscle satellite cells of one-week old mice were activated, while most of the muscle satellite cells of adult mice were in resting state. Real-time quantitative PCR analysis showed that KLF7 was highly expressed in resting muscle satellite cells, and protein expression analysis confirmed the similar expression trend. The high expression level of KLF7 in non-proliferative cells interferes with KLF7 and promotes the activation, expansion and proliferation of muscle satellite cells. Overexpression of KLF7 promotes myoblast cell cycle arrest, indicating that KLF7 is the key factor to regulate resting of muscle satellite cells.) KLF7 regulates the expression of p21 in myoblasts. Interference p21 inhibited the activation and expansion of muscle satellite cells by KLF7. The regulation of KLF7 on the proliferation and p21 expression of C2C12 myoblasts was inhibited, which was confirmed by double luciferase report. The mutation of acetylated site resulted in a significant decrease in the regulation of p21 promoter by KLF7, indicating that KLF7 acetylated site Lys227 and / or Lys231. TGF- 尾 signaling pathway negatively regulates the expression of KLF7 in myoblasts and interferes with KLF7. TGF- 尾 signaling pathway block induced muscle satellite cell resting and partial loss of myoblast proliferation inhibition. Supporting KLF7 is an essential factor of blocking TGF- 尾 signaling pathway to induce muscle satellite cells to rest.) the expression of KLF7 is up-regulated in muscle cells by activating classical Notch signaling pathway. Interference with KLF7 at least partially removes the activation of classical Notch signaling pathway that induces the proliferation inhibition of muscle cells. The results showed that KLF7 mediated the classical Notch signaling pathway and interfered with the expression of myoblast differentiation factor and marker gene by interfering with KLF7. It is suggested that KLF7 may not be involved in the regulation of myogenic differentiation. By analyzing the above results, we can draw the following conclusions: 1 / 1 KLF7 plays a key role in the resting regulation of muscle satellite cells. Inhibition of myoblast cell cycle and regulation of muscle cell activation and proliferation by KLF7 were dependent on cell cycle inhibitor p21.3). The Lys227 and Lys231 sites of KLF7 protein. It may be that acetylated modified KLF7 mediates the regulation of extracellular signal (classical TGF- 尾 and Notch signaling pathway) on the resting of muscle satellite cells.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.2

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