巨噬细胞剔除对损伤骨骼肌再生的影响及机制研究

发布时间：2018-03-22 00:13

  本文选题：骨骼肌　切入点：损伤修复　出处：《上海体育学院》2017年硕士论文　论文类型：学位论文

【摘要】：研究目的:骨骼肌损伤是最常见,最棘手的运动损伤之一。骨骼肌损伤后巨噬细胞在其修复过程中发挥重要作用。但目前对骨骼肌损伤后巨噬细胞发挥作用的机制并不明了。因此,我们建立小鼠骨骼肌钝挫伤模型和巨噬细胞剔除模型,观察巨噬细胞剔除后骨骼肌损伤修复过程中肌卫星细胞增殖分化、炎症因子、肌再生调节因子、趋化因子、血管再生因子、氧化应激因子表达规律及Akt/mTOR信号通路激活状况,以深入探究巨噬细胞在骨骼肌损伤修复中的作用及其机制。研究方法:80只雄性C57BL/6小鼠随机分为骨骼肌损伤组(S,n=32),未损伤对照组(SConn,n=8),损伤+巨噬细胞剔除组(T,n=32),未损伤+巨噬细胞剔除对照组(TCon,n=8)。骨骼肌钝挫伤后1d,3d,7d和14d取双侧腓肠肌。流式细胞术检测巨噬细胞剔除效果,HE染色观察骨骼肌形态学变化,荧光定量PCR及western blotting检测炎症因子、肌再生因子、趋化因子、血管再生因子、氧化应激因子、及Akt/mTOR蛋白质合成信号分子表达变化。研究结果:1.巨噬细胞剔除损害骨骼肌再生HE染色结果显示,骨骼肌钝挫伤后肌纤维结构破坏、大量肌纤维坏死、肿胀(损伤第1d,3d),损伤第7d出现大量再生肌纤维,而剔除组在伤后第7d仅出现少量再生肌纤维。伤后第14d巨噬细胞剔除组仍有大量再生肌纤维出现,提示巨噬细胞剔除损害骨骼肌再生。2.巨噬细胞剔除对肌卫星细胞增殖分化的影响荧光定量PCR结果显示,骨骼肌损伤后第1d和3d肌卫星细胞增殖标志物MyoD和分化标志物myogenin表达均显著高于对照组(p0.01)。而与损伤组相比,巨噬细胞剔除显著抑制了 MyoD在伤后第7d的表达(p0.05),促进myogenin在伤后第14d的表达(p0.05)。3.巨噬细胞剔除对肌再生因子表达的影响损伤组HGF mRNA在伤后第1d,3d和7d表达量均显著增加(p0.01),巨噬细胞剔除组HGF在伤后第1d和3d表达量显著低于损伤组(p0.05)。uPA和IGF-1mRNA的表达与HGF相似,在损伤后表达量显著增加,巨噬细胞剔除后表达显著降低。MGF在伤后表达无显著变化,但巨噬细胞剔除显著下调其在伤后第1d和7d的表达。损伤组GDF11 mRNA在伤后第1d表达显著增加,巨噬细胞剔除组GDF11亦在伤后第1d表达显著增加,且与损伤组相比在伤后第7d的表达显著降低(p0.05)。损伤组CB2R mRNA在伤后1d和3d表达显著增加(p0.01),而与损伤组相比巨噬细胞剔除显著抑制其在伤后1d和7d的表达。4.巨噬细胞剔除对损伤骨骼肌炎症因子表达的影响促炎细胞因子TNF-α mRNA在伤后第1d,3d,7d和14d表达显著增加(p0.01)。与损伤组相比,巨噬细胞剔除显著提高TNF-αmRNA在伤后第7d的表达(p0.01),抑制其在伤后1d的表达(p0.05)。IFN-γ和IL-6mRNA的表达与TNF-α表达相似,在伤后表达显著增加,巨噬细胞剔除显著促进其在伤后第14d表达(p0.05)。损伤组IL-12mRNA在伤后1d和3d表达显著增加(p0.01),巨噬细胞剔除组仅在伤后3d表达显著增加(p0.01)。而与损伤组相比,巨噬细胞剔除组中IL-12 mRNA表达并无显著变化。损伤组TWEAK mRNA在伤后3d和7d表达均显著增加,剔除组TWEAK mRNA在伤后1d和7d表达显著下降,且剔除组TWEAK mRNA在伤后1d和7d表达显著低于损伤组,而在损伤前及伤后第14d表达均显著高于损伤组。抑炎细胞因子IL-10 mRNA在伤后1-7d表达量显著增加(p0.01),而巨噬细胞剔除显著下调其在伤后第1d的表达(p0.05)。5.巨噬细胞剔除对损伤骨骼肌趋化因子表达的影响RT-PCR结果显示,损伤组中骨骼肌趋化因子(CCL2,CCL3,CCL8,CXCR4)mRNA在伤后1-7d表达均显著增加,而CCL4mRNA在伤后第14d表达显著降低(p0.01),CXCL12仅在伤后第7d表达显著增加(p0.01)。与损伤组相比,巨噬细胞剔除显著提高CCL2在伤后第14d表达(p0.05),显著提高CCL3在伤后3d和14d的表达(p0.05),下调CCL4在伤后第1d表达(p0.01),提高CCL4在伤后第14d表达(p0.01),下调CCL8在伤后1d和3d表达(p0.01),提高伤后第14d表达(p0.01),上调CXCL12和CXCR4在伤后14d表达(p0.01)。6.巨噬细胞剔除对损伤骨骼肌氧化应激因子表达的影响RT-PCR结果显示,gp91phox mRNA在伤后1d,3d和7d表达均显著增加,剔除组gp91phox mRNA在伤后7d和14d表达均显著增加(p0.01),且与损伤组相比在伤后第1d,3d和7d表达显著下降,伤后14d表达显著增加(p0.05)。损伤组gp91phox蛋白在伤后第3d表达显著增加(p0.01)。巨噬细胞剔除组gP91phox蛋白表达与剔除对照组相比并无显著差异。但在伤后第14d,剔除组gp91phox蛋白表达显著高于损伤组(p0.01)。7.巨噬细胞剔除对损伤骨骼肌血管再生因子表达的影响损伤组HIF-1α mRNA在伤后1d,3d和7d表达均显著增加,剔除组损伤前及伤后第14d HIF-1α mRNA表达显著高于损伤组。损伤组Angpt1在伤后1d,3d,7d,14d表达均显著增加(p0.01)。剔除组Angpt1 伤后1-14d表达均显著增加(p0.01),且在损伤前以及损伤后第14d表达均显著高于损伤组(p0.05)。损伤组VEGF在伤后第7d表达显著低于对照组(p0.05),剔除组VEGFmRNA在伤后1-14d表达均显著降低(p0.01),而剔除对照组VEGF mRNA显著高于损伤对照组(p0.05)。8.巨噬细胞剔除对损伤骨骼肌蛋白质合成信号分子表达的影响Western Blotting结果显示,损伤组骨骼肌p-Akt/Akt和p-mTOR/mTOR均在伤后第1d表达显著增加(p0.05),p-p70s6k/p70s6k在伤后1d和3d表达均显著增加,P-4EBP1/4EBP1在伤后1d,3d,7d和14d表达均显著增加。而巨噬细胞剔除组 p-Akt/Akt,p-mTOR/mTOR,p-p70s6k/p70s6k 和 P-4EBP1/4EBP1 与对照组及损伤组相比虽有增加,但并无显著变化(p0.05)。研究结论:1.骨骼肌损伤修复过程中肌卫星细胞增殖分化标志物、巨噬细胞、炎症因子、肌再生因子、趋化因子、血管再生因子、氧化应激因子以及蛋白质合成信号分子表达均出现显著变化,提示其可能在骨骼肌损伤修复过程中发挥重要作用。2.巨噬细胞剔除可损害骨骼肌损伤再生,其机制可能与下调肌再生因子表达、上调损伤后期炎症因子、趋化因子、氧化应激水平,抑制血管再生以及Akt/mTOR蛋白质合成信号通路的激活有关,巨噬细胞在损伤骨骼肌修复过程中发挥了重要作用。
[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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