本文關(guān)鍵詞：KLF7基因調(diào)控肌肉衛(wèi)星細(xì)胞靜息的分子機(jī)制研究　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： KLF7 肌肉衛(wèi)星細(xì)胞 靜息 TGF-β Notch

【摘要】：骨骼肌具有強大的再生能力,肌肉損傷后需要募集前體干細(xì)胞到受傷部位,完成肌纖維的修復(fù)和生成,在成年骨骼肌中,肌肉衛(wèi)星細(xì)胞行使此功能。肌肉衛(wèi)星細(xì)胞是特異性肌肉干細(xì)胞,因其解剖位置位于肌纖維膜和基底膜之間而得名。在健康的成年動物中,肌肉衛(wèi)星細(xì)胞維持靜息狀態(tài),當(dāng)受到生理或病理刺激下,能夠快速激活,進(jìn)行擴(kuò)增及不對稱分裂,完成自我更新,形成新肌纖維。維持肌肉衛(wèi)星細(xì)胞靜息態(tài)對于保持肌肉干細(xì)胞群數(shù)量及再生潛能意義重大。越來越多證據(jù)表明,多種胞外信號參與肌肉衛(wèi)星細(xì)胞靜息調(diào)節(jié),當(dāng)靜息態(tài)調(diào)控因子失活或信號通路被阻斷時,肌肉衛(wèi)星細(xì)胞生理功能發(fā)生嚴(yán)重紊亂,肌肉再生能力顯著下降。Krüppel家族(KLFs)多種因子參與肌肉細(xì)胞生理調(diào)控,而KLF7在神經(jīng)發(fā)育及髓細(xì)胞增殖中發(fā)揮重要調(diào)節(jié)作用,表達(dá)于骨骼肌及成肌細(xì)胞,但其調(diào)控作用及機(jī)制還未探明。本研究通過體外培養(yǎng)小鼠肌肉衛(wèi)星細(xì)胞及單根肌纖維,深入分析KLF7在肌肉細(xì)胞的調(diào)節(jié)功能。試驗結(jié)果發(fā)現(xiàn):(1)一周齡小鼠肌肉衛(wèi)星細(xì)胞多數(shù)為激活態(tài),而成年小鼠肌肉衛(wèi)星細(xì)胞大多處于靜息態(tài),實時定量PCR分析發(fā)現(xiàn)KLF7在靜息態(tài)肌肉衛(wèi)星細(xì)胞中表達(dá)較高,蛋白表達(dá)分析證實了相似的表達(dá)趨勢。在成肌細(xì)胞的研究顯示,KLF7在非增殖細(xì)胞中,表達(dá)水平較高。(2)干擾KLF7促進(jìn)肌肉衛(wèi)星細(xì)胞激活、擴(kuò)增,以及成肌細(xì)胞增殖,而過表達(dá)KLF7促進(jìn)成肌細(xì)胞細(xì)胞周期阻滯,表明KLF7是調(diào)控肌肉衛(wèi)星細(xì)胞靜息的關(guān)鍵因子。(3)成肌細(xì)胞中,KLF7調(diào)控p21的表達(dá),而干擾p21抑制KLF7對肌肉衛(wèi)星細(xì)胞激活和擴(kuò)增的調(diào)控作用。(4)乙�；揎椢稽c突變后,KLF7對C2C12成肌細(xì)胞增殖和p21表達(dá)的調(diào)控作用受到抑制,雙熒光素酶報告分析證實,乙�；稽c突變導(dǎo)致KLF7對p21啟動子的調(diào)控顯著降低,表明KLF7乙�；稽cLys227和/或Lys231,對其在成肌細(xì)胞調(diào)控中起重要作用。(5)成肌細(xì)胞中,TGF-β信號通路負(fù)調(diào)控KLF7的表達(dá),干擾KLF7后,TGF-β信號通路阻斷誘導(dǎo)的肌肉衛(wèi)星細(xì)胞靜息和成肌細(xì)胞增殖抑制部分喪失,支持KLF7是TGF-β信號通路阻斷誘導(dǎo)肌肉衛(wèi)星細(xì)胞靜息的必需因子。(6)肌肉細(xì)胞中激活經(jīng)典Notch信號通路,KLF7表達(dá)上調(diào),而干擾KLF7至少部分移除經(jīng)典Notch信號通路激活誘導(dǎo)的肌肉細(xì)胞細(xì)胞增殖抑制,表明KLF7介導(dǎo)了經(jīng)典Notch信號通路調(diào)節(jié)的肌肉細(xì)胞靜息。(7)干擾KLF7對成肌細(xì)胞成肌分化因子和標(biāo)記基因表達(dá)沒有影響,暗示KLF7可能沒有參與成肌分化調(diào)控。綜合分析上述試驗結(jié)果,得出以下結(jié)論:1)KLF7在肌肉衛(wèi)星細(xì)胞靜息調(diào)控中起關(guān)鍵作用,并抑制成肌細(xì)胞細(xì)胞周期進(jìn)行。2)KLF7調(diào)控肌肉細(xì)胞激活和增殖依賴于細(xì)胞周期抑制因子p21。3)KLF7蛋白的Lys227和Lys231位點,對自身功能激活非常重要,可能存在乙�；揎�。4)KLF7介導(dǎo)了胞外信號(經(jīng)典TGF-β和Notch信號通路)對肌肉衛(wèi)星細(xì)胞靜息的調(diào)節(jié)。
[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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.2

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