發(fā)布時(shí)間：2018-03-22 00:13

  本文選題：骨骼肌　切入點(diǎn)：損傷修復(fù)　出處：《上海體育學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的:骨骼肌損傷是最常見,最棘手的運(yùn)動損傷之一。骨骼肌損傷后巨噬細(xì)胞在其修復(fù)過程中發(fā)揮重要作用。但目前對骨骼肌損傷后巨噬細(xì)胞發(fā)揮作用的機(jī)制并不明了。因此,我們建立小鼠骨骼肌鈍挫傷模型和巨噬細(xì)胞剔除模型,觀察巨噬細(xì)胞剔除后骨骼肌損傷修復(fù)過程中肌衛(wèi)星細(xì)胞增殖分化、炎癥因子、肌再生調(diào)節(jié)因子、趨化因子、血管再生因子、氧化應(yīng)激因子表達(dá)規(guī)律及Akt/mTOR信號通路激活狀況,以深入探究巨噬細(xì)胞在骨骼肌損傷修復(fù)中的作用及其機(jī)制。研究方法:80只雄性C57BL/6小鼠隨機(jī)分為骨骼肌損傷組(S,n=32),未損傷對照組(SConn,n=8),損傷+巨噬細(xì)胞剔除組(T,n=32),未損傷+巨噬細(xì)胞剔除對照組(TCon,n=8)。骨骼肌鈍挫傷后1d,3d,7d和14d取雙側(cè)腓腸肌。流式細(xì)胞術(shù)檢測巨噬細(xì)胞剔除效果,HE染色觀察骨骼肌形態(tài)學(xué)變化,熒光定量PCR及western blotting檢測炎癥因子、肌再生因子、趨化因子、血管再生因子、氧化應(yīng)激因子、及Akt/mTOR蛋白質(zhì)合成信號分子表達(dá)變化。研究結(jié)果:1.巨噬細(xì)胞剔除損害骨骼肌再生HE染色結(jié)果顯示,骨骼肌鈍挫傷后肌纖維結(jié)構(gòu)破壞、大量肌纖維壞死、腫脹(損傷第1d,3d),損傷第7d出現(xiàn)大量再生肌纖維,而剔除組在傷后第7d僅出現(xiàn)少量再生肌纖維。傷后第14d巨噬細(xì)胞剔除組仍有大量再生肌纖維出現(xiàn),提示巨噬細(xì)胞剔除損害骨骼肌再生。2.巨噬細(xì)胞剔除對肌衛(wèi)星細(xì)胞增殖分化的影響熒光定量PCR結(jié)果顯示,骨骼肌損傷后第1d和3d肌衛(wèi)星細(xì)胞增殖標(biāo)志物MyoD和分化標(biāo)志物myogenin表達(dá)均顯著高于對照組(p0.01)。而與損傷組相比,巨噬細(xì)胞剔除顯著抑制了 MyoD在傷后第7d的表達(dá)(p0.05),促進(jìn)myogenin在傷后第14d的表達(dá)(p0.05)。3.巨噬細(xì)胞剔除對肌再生因子表達(dá)的影響損傷組HGF mRNA在傷后第1d,3d和7d表達(dá)量均顯著增加(p0.01),巨噬細(xì)胞剔除組HGF在傷后第1d和3d表達(dá)量顯著低于損傷組(p0.05)。uPA和IGF-1mRNA的表達(dá)與HGF相似,在損傷后表達(dá)量顯著增加,巨噬細(xì)胞剔除后表達(dá)顯著降低。MGF在傷后表達(dá)無顯著變化,但巨噬細(xì)胞剔除顯著下調(diào)其在傷后第1d和7d的表達(dá)。損傷組GDF11 mRNA在傷后第1d表達(dá)顯著增加,巨噬細(xì)胞剔除組GDF11亦在傷后第1d表達(dá)顯著增加,且與損傷組相比在傷后第7d的表達(dá)顯著降低(p0.05)。損傷組CB2R mRNA在傷后1d和3d表達(dá)顯著增加(p0.01),而與損傷組相比巨噬細(xì)胞剔除顯著抑制其在傷后1d和7d的表達(dá)。4.巨噬細(xì)胞剔除對損傷骨骼肌炎癥因子表達(dá)的影響促炎細(xì)胞因子TNF-α mRNA在傷后第1d,3d,7d和14d表達(dá)顯著增加(p0.01)。與損傷組相比,巨噬細(xì)胞剔除顯著提高TNF-αmRNA在傷后第7d的表達(dá)(p0.01),抑制其在傷后1d的表達(dá)(p0.05)。IFN-γ和IL-6mRNA的表達(dá)與TNF-α表達(dá)相似,在傷后表達(dá)顯著增加,巨噬細(xì)胞剔除顯著促進(jìn)其在傷后第14d表達(dá)(p0.05)。損傷組IL-12mRNA在傷后1d和3d表達(dá)顯著增加(p0.01),巨噬細(xì)胞剔除組僅在傷后3d表達(dá)顯著增加(p0.01)。而與損傷組相比,巨噬細(xì)胞剔除組中IL-12 mRNA表達(dá)并無顯著變化。損傷組TWEAK mRNA在傷后3d和7d表達(dá)均顯著增加,剔除組TWEAK mRNA在傷后1d和7d表達(dá)顯著下降,且剔除組TWEAK mRNA在傷后1d和7d表達(dá)顯著低于損傷組,而在損傷前及傷后第14d表達(dá)均顯著高于損傷組。抑炎細(xì)胞因子IL-10 mRNA在傷后1-7d表達(dá)量顯著增加(p0.01),而巨噬細(xì)胞剔除顯著下調(diào)其在傷后第1d的表達(dá)(p0.05)。5.巨噬細(xì)胞剔除對損傷骨骼肌趨化因子表達(dá)的影響RT-PCR結(jié)果顯示,損傷組中骨骼肌趨化因子(CCL2,CCL3,CCL8,CXCR4)mRNA在傷后1-7d表達(dá)均顯著增加,而CCL4mRNA在傷后第14d表達(dá)顯著降低(p0.01),CXCL12僅在傷后第7d表達(dá)顯著增加(p0.01)。與損傷組相比,巨噬細(xì)胞剔除顯著提高CCL2在傷后第14d表達(dá)(p0.05),顯著提高CCL3在傷后3d和14d的表達(dá)(p0.05),下調(diào)CCL4在傷后第1d表達(dá)(p0.01),提高CCL4在傷后第14d表達(dá)(p0.01),下調(diào)CCL8在傷后1d和3d表達(dá)(p0.01),提高傷后第14d表達(dá)(p0.01),上調(diào)CXCL12和CXCR4在傷后14d表達(dá)(p0.01)。6.巨噬細(xì)胞剔除對損傷骨骼肌氧化應(yīng)激因子表達(dá)的影響RT-PCR結(jié)果顯示,gp91phox mRNA在傷后1d,3d和7d表達(dá)均顯著增加,剔除組gp91phox mRNA在傷后7d和14d表達(dá)均顯著增加(p0.01),且與損傷組相比在傷后第1d,3d和7d表達(dá)顯著下降,傷后14d表達(dá)顯著增加(p0.05)。損傷組gp91phox蛋白在傷后第3d表達(dá)顯著增加(p0.01)。巨噬細(xì)胞剔除組gP91phox蛋白表達(dá)與剔除對照組相比并無顯著差異。但在傷后第14d,剔除組gp91phox蛋白表達(dá)顯著高于損傷組(p0.01)。7.巨噬細(xì)胞剔除對損傷骨骼肌血管再生因子表達(dá)的影響損傷組HIF-1α mRNA在傷后1d,3d和7d表達(dá)均顯著增加,剔除組損傷前及傷后第14d HIF-1α mRNA表達(dá)顯著高于損傷組。損傷組Angpt1在傷后1d,3d,7d,14d表達(dá)均顯著增加(p0.01)。剔除組Angpt1 傷后1-14d表達(dá)均顯著增加(p0.01),且在損傷前以及損傷后第14d表達(dá)均顯著高于損傷組(p0.05)。損傷組VEGF在傷后第7d表達(dá)顯著低于對照組(p0.05),剔除組VEGFmRNA在傷后1-14d表達(dá)均顯著降低(p0.01),而剔除對照組VEGF mRNA顯著高于損傷對照組(p0.05)。8.巨噬細(xì)胞剔除對損傷骨骼肌蛋白質(zhì)合成信號分子表達(dá)的影響Western Blotting結(jié)果顯示,損傷組骨骼肌p-Akt/Akt和p-mTOR/mTOR均在傷后第1d表達(dá)顯著增加(p0.05),p-p70s6k/p70s6k在傷后1d和3d表達(dá)均顯著增加,P-4EBP1/4EBP1在傷后1d,3d,7d和14d表達(dá)均顯著增加。而巨噬細(xì)胞剔除組 p-Akt/Akt,p-mTOR/mTOR,p-p70s6k/p70s6k 和 P-4EBP1/4EBP1 與對照組及損傷組相比雖有增加,但并無顯著變化(p0.05)。研究結(jié)論:1.骨骼肌損傷修復(fù)過程中肌衛(wèi)星細(xì)胞增殖分化標(biāo)志物、巨噬細(xì)胞、炎癥因子、肌再生因子、趨化因子、血管再生因子、氧化應(yīng)激因子以及蛋白質(zhì)合成信號分子表達(dá)均出現(xiàn)顯著變化,提示其可能在骨骼肌損傷修復(fù)過程中發(fā)揮重要作用。2.巨噬細(xì)胞剔除可損害骨骼肌損傷再生,其機(jī)制可能與下調(diào)肌再生因子表達(dá)、上調(diào)損傷后期炎癥因子、趨化因子、氧化應(yīng)激水平,抑制血管再生以及Akt/mTOR蛋白質(zhì)合成信號通路的激活有關(guān),巨噬細(xì)胞在損傷骨骼肌修復(fù)過程中發(fā)揮了重要作用。
[Abstract]:Objective: To study the injury of skeletal muscle is the most common, one of the most difficult sports injury. The injury of skeletal muscle after macrophages in the repair process play an important role. But the skeletal muscle injury of macrophages play a role in the mechanism is not clear. Therefore, we established mouse skeletal muscle contusion model and macrophage elimination model, muscle satellite to observe the proliferation and differentiation of cells, macrophages removed after the injury and repair of skeletal muscle during inflammation, regulator of muscle regeneration, chemokines, angiogenesis, expression and activation of Akt/mTOR signal pathway of oxidative stress factors, to delve into macrophages in skeletal muscle injury in rats and its mechanism. Methods: 80 male C57BL/6 mice were randomly divided into skeletal muscle injury group (S, n=32), non injury group (SConn, n=8), injury group (T + macrophages, excluding n=32), without injury + macrophage Excluding the cell control group (TCon, n=8). Contusion of skeletal muscle after 1D, 3D, 7d and 14d in gastrocnemius muscles. Excluding the effect of macrophages were detected by flow cytometry, observe the morphological changes of skeletal muscle inflammatory factor HE staining, fluorescence quantitative PCR detection of Western and blotting, muscle regeneration factor, chemotactic factor, angiogenesis factor, oxidative stress factor, Akt/mTOR expression and protein synthesis of signal molecules. Results: 1. macrophages eliminate damage of skeletal muscle regeneration HE staining showed that the damage of muscle fiber structure of skeletal muscle after contusion, a lot of muscle fiber necrosis, swelling (injury 1D, 3D, 7D) the emergence of a large number of injury and regeneration of muscle fiber. And eliminate group after injury 7d only small muscle fiber regeneration after injury. The 14d group is still a large number of macrophages from regeneration of muscle fibers, suggesting that macrophages from damage of skeletal muscle regeneration in.2. macrophage cells to remove fine muscle satellite Influence of fluorescent quantitative PCR results showed cell proliferation and differentiation, skeletal muscle injury after 1D and 3D muscle satellite cell proliferation marker MyoD and differentiation marker myogenin expression were significantly higher than that of the control group (P0.01). Compared with the injury group, macrophage elimination significantly inhibited the expression of MyoD in the 7d after injury (P0.05). Promote the expression of myogenin in the 14d after injury (P0.05) in.3. macrophages to remove muscle regeneration effect to the expression of HGF mRNA in injury group after injury 1D, 3D and 7d expression were significantly increased (P0.01), HGF group was significantly lower than that in macrophages from the 1D and 3D expression after injury injury group (P0.05) the expression of HGF and.UPA and IGF-1mRNA were similar, significant increase in expression after injury, macrophage after excluding expression decreased significantly at.MGF after injury expression did not change significantly, but excluding the macrophages significantly down regulated after injury in 1D and 7d expression of GDF11 mRNA in injury group. A significant increase in the expression of 1D in macrophages after injury, excluding group GDF11 also increased significantly in the expression of 1D after injury, and the injury group were significantly decreased in the expression of 7D after injury (P0.05). Compared with the injury group CB2R at mRNA after injury the expression of 1D and 3D increased significantly (P0.01), and compared with the injury group effect the expression of.4. in macrophages macrophages inhibit the elimination removed at 1D after injury and the expression of 7D on skeletal muscle injury inflammatory factor of proinflammatory cytokine TNF- alpha mRNA after injury 1D, 3D, 7d and 14d expression increased significantly (P0.01). Compared with the injury group, macrophage elimination significantly increased expression of mRNA in TNF- the 7d (P0.01) after injury, inhibiting its expression after injury 1D (P0.05) expression and TNF- expression of.IFN- gamma and IL-6mRNA similar expression was significantly increased after injury, macrophage elimination significantly promote its expression in the 14d after injury (P0.05). At 1D after injury and 3D injury group IL-12mRNA A significant increase (P0.01), macrophage elimination group only after injury 3D expression increased significantly (P0.01). Compared with the injury group, there was no significant change of IL-12 expression in mRNA macrophage elimination group. At 3D after injury and 7d expression were significantly increased in injury group TWEAK mRNA group TWEAK, excluding mRNA in 1D and 7d after injury. The expression of TWEAK group decreased significantly, and eliminate the mRNA at 1D after injury and the expression of 7D was significantly lower than that in the injury group, while in the injury before and after injury the expression of 14d was significantly higher than that in the injury group. Anti inflammatory cytokines IL-10 mRNA after injury 1-7d expression was significantly increased (P0.01), and macrophage elimination significantly suppressed in the expression of 1D after injury (P0.05) in.5. macrophages to remove the muscle injury effect of chemokine RT-PCR expression results showed that skeletal muscle chemokine injury group (CCL2, CCL3, CCL8, CXCR4) mRNA expression of 1-7d after injury were significantly increased, while the CCL4mRNA after the injury in the 14d table 杈炬樉钁楅檷浣，

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