骨骼肌挫伤修复过程中巨噬细胞的趋化机制
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摘要
目的:探究巨噬细胞在挫伤骨骼肌修复过程中的趋化机制。方法:将66只ICR雄性小鼠随机分为对照组(C组,n=11)和骨骼肌挫伤组(S组,n=55)。骨骼肌挫伤后12 h、1 d、5 d、10 d和15 d分别取双侧腓肠肌。HE染色观察骨骼肌挫伤后修复过程中的形态学变化,荧光定量PCR检测骨骼肌挫伤后巨噬细胞和趋化因子的时空表达规律。结果:①骨骼肌挫伤后12 h,可见肌纤维坏死、肿胀和大量炎性细胞浸润。伤后第5天,可见少量再生肌纤维,伤后第10天再生肌纤维数量大大增加,伤后第15天虽有少量再生肌纤维,但肌纤维结构完整性较好,骨骼肌损伤修复基本完成。②与对照组相比,M1巨噬细胞标志物(CD68)在骨骼肌挫伤后显著增加,随后M2巨噬细胞标志物(CD206和CD163)显著增加。③骨骼肌挫伤后MCP-1及其受体CCR2 mRNA表达均显著增加(P<0.01)。SDF-1 mRNA在挫伤后12 h、10 d和15 d表达均显著增加(P<0.01),其受体CXCR4表达无显著变化。④骨骼肌挫伤后,MCP-1与CD68存在强相关,亦与其受体CCR2呈强相关,而SDF-1只与其受体CXCR4相关。结论:MCP-1/CCR2轴可能参与了挫伤骨骼肌修复过程中M1巨噬细胞的趋化。
Objective:The objective of this study is to explore the mechanism of macrophage chemotaxis during the repair process following contusion of skeletal muscle.Methods:66 ICR male mice were randomly divided into control group(C group,n=11) and muscle contusion group(S group,n=55).Their gastrocnemius muscles were harvested at the time points of 12 h,1 d,5 d,10 d and 15 d post-injury. The changes in skeletal muscle morphology were assessed by HE staining. The gene expression of macrophage and chemokines was analyzed by real-time PCR.Results:① On 12 h post-injury, cross sections of gastrocnemius muscles showed substantial fiber damage and inflammatory infiltrate.On day 5 post-injury,a small quantity of centronucleated myofibers were observed.On day 10 post-injury,central nucleation phenomenon became more pronounced.On day 15 post-injury,central nucleation almost disappeared.② The molecule marker of M1 macrophages(CD68) increased significantly post-injury,and the molecule marker of M2 macrophages(CD206 and CD163) increased significantly later.③ MCP-1 and CCR2 mRNA increased significantly post-injury(P<0.01).SDF-1 mRNA increased significantly at 12 h,10 d and 15 d(P<0.01).However,CXCR4 mRNA did not change significantly post injury.④ The results showed that there was significant correlation between MCP-1 and CD68,and CCR2.In addition,there was significant correlation between SDF-1 and CXCR4.Conclusion:MCP-1/CCR2 may be involved in the chemotaxis of M1 macrophages during skeletal muscle regeneration post-injury.
Objective:The objective of this study is to explore the mechanism of macrophage chemotaxis during the repair process following contusion of skeletal muscle.Methods:66 ICR male mice were randomly divided into control group(C group,n=11) and muscle contusion group(S group,n=55).Their gastrocnemius muscles were harvested at the time points of 12 h,1 d,5 d,10 d and 15 d post-injury. The changes in skeletal muscle morphology were assessed by HE staining. The gene expression of macrophage and chemokines was analyzed by real-time PCR.Results:① On 12 h post-injury, cross sections of gastrocnemius muscles showed substantial fiber damage and inflammatory infiltrate.On day 5 post-injury,a small quantity of centronucleated myofibers were observed.On day 10 post-injury,central nucleation phenomenon became more pronounced.On day 15 post-injury,central nucleation almost disappeared.② The molecule marker of M1 macrophages(CD68) increased significantly post-injury,and the molecule marker of M2 macrophages(CD206 and CD163) increased significantly later.③ MCP-1 and CCR2 mRNA increased significantly post-injury(P<0.01).SDF-1 mRNA increased significantly at 12 h,10 d and 15 d(P<0.01).However,CXCR4 mRNA did not change significantly post injury.④ The results showed that there was significant correlation between MCP-1 and CD68,and CCR2.In addition,there was significant correlation between SDF-1 and CXCR4.Conclusion:MCP-1/CCR2 may be involved in the chemotaxis of M1 macrophages during skeletal muscle regeneration post-injury.
引文
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[23] ZOU K,WANG Y,HU Y,et al.Specific tumor-derived CCL2 mediated by pyruvate kinase M2 in colorectal cancer cells contributes to macrophage recruitment in tumor microenvironment[J].Tumour Biol,2017,39(3):1-10
[24] 杨建斌,冯红超,宋宇峰.口腔鳞癌中单核细胞趋化蛋白-1在巨噬细胞浸润、聚集中的意义 [J].中国肿瘤临床,2005,32(20):1155-1157
[25] CARSON W F T,SALTER G S E,SCOLA M M,et al.Enhancement of macrophage inflammatory responses by CCL2 is correlated with increased miR-9 expression and downregulation of the ERK1/2 phosphatase Dusp6 [J].Cell Immunol,2017,314:63-72
[26] CONTRERAS S V,OCHOA O,REYES R S M,et al.Fat accumulation with altered inflammation and regeneration in skeletal muscle of CCR2-/- mice following ischemic injury[J].American Journal of Physiology Cell Physiology,2007,292(2):C953-967
[27] OCHOA O,SUN D,REYES R S M,et al.Delayed angiogenesis and VEGF production in CCR2-/- mice during impaired skeletal muscle regeneration [J].American Journal of Physiology Regulatory,Integrative and Comparative Physiology,2007,293(2):R651-661
[28] ARNOLD L,HENRY A,PORON F,et al.Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis [J].J Exp Med,2007,204(5):1057-1069
[29] BRZOSKA E,KOWALSKI K,MARKOWSKA Z A,et al.Sdf-1 (CXCL12) induces CD9 expression in stem cells engaged in muscle regeneration[J].Stem Cell Research & Therapy,2015,6:46
[30] BRZOSKA E,KOWALEWSKA M,MARKOWSKA Z A,et al.Sdf-1 (CXCL12) improves skeletal muscle regeneration via the mobilisation of Cxcr4 and CD34 expressing cells[J].Biology of The Cell/Under The Auspices of The European Cell Biology Organization,2012,104(12):722-737
[31] RATAJCZAK M Z,MAJKA M,KUCIA M,et al.Expression of functional CXCR4 by muscle satellite cells and secretion of SDF-1 by muscle-derived fibroblasts is associated with the presence of both muscle progenitors in bone marrow and hematopoietic stem/progenitor cells in muscles[J].Stem Cells,2003,21(3):363-371
[32] 杨志峰,杨清玲,陈昌杰.趋化因子SDF-1与受体CXCR4的研究进展 [J].分子诊断与治疗杂志,2011,3(1):58-61
[33] 李鹏程,徐茜,胡燕华.蛋白激酶C对SDF-1诱导巨噬细胞趋化及血管内皮生长因子转录的调节作用 [J].华中科技大学学报(医学版),2008,37(1):93-96
[2] RELAIX F,ZAMMIT P S.Satellite cells are essential for skeletal muscle regeneration:The cell on the edge returns centre stage [J].Development,2012,139(16):2845-2856
[3] TIDBALL J G,VILLALTA S A.Regulatory interactions between muscle and the immune system during muscle regeneration [J].American Journal of Physiology Regulatory,Integrative and Comparative Physiology,2010,298(5):R1173-1187
[4] KHARRAZ Y,GUERRA J,MANN C J,et al.Macrophage plasticity and the role of inflammation in skeletal muscle repair [J].Mediators of Inflammation,2013,2013:491-497
[5] MARTINEZ C O,MCHALE M J,WELLS J T,et al.Regulation of skeletal muscle regeneration by CCR2-activating chemokines is directly related to macrophage recruitment [J].American Journal of Physiology Regulatory,Integrative and Comparative Physiology,2010,299(3):R832-842
[6] SHIREMAN P K,CONTRERAS S V,OCHOA O,et al.MCP-1 deficiency causes altered inflammation with impaired skeletal muscle regeneration[J].Journal of Leukocyte Biology,2007,81(3):775-785
[7] XIAO W,LIU Y,LUO B,et al.Time-dependent gene expression analysis after mouse skeletal muscle contusion[J].Journal of Sport and Health Science,2016,5(1):101-108
[8] XIAO W,LIU Y,CHEN P.Macrophage depletion impairs skeletal muscle regeneration:The roles of pro-fibrotic factors,inflammation,and oxidative stress[J].Inflammation,2016,39(6):2016-2028
[9] LIU X,LIU Y,ZHAO L,et al.Macrophage depletion impairs skeletal muscle regeneration:The roles of regulatory factors for muscle regeneration[J].Cell Biology International,2017,41(3):228-238
[10] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method[J].Methods,2001,25(4):402-408
[11] XIAO W,CHEN P,DONG J.Effects of overtraining on skeletal muscle growth and gene expression[J].International Journal of Sports Medicine,2012,33(10):846-853
[12] 肖卫华,陈佩杰,刘宇.巨噬细胞在骨骼肌急性损伤修复中的作用研究进展[J].中国运动医学杂志,2014,33(3):269-274
[13] CEAFALAN L C,MANOLE E,TANASE C P,et al.Interstitial outburst of angiogenic factors during skeletal muscle regeneration after acute mechanical trauma[J].Anat Rec (Hoboken),2015,298(11):1864-1879
[14] WRIGHT C T,OPOLON P,APPELL H J,et al.Treatment of muscle injuries by local administration of autologous conditioned serum:Animal experiments using a muscle contusion model[J].International Journal of Sports Medicine,2004,25(8):582-587
[15] NOZAKI M,LI Y,ZHU J,et al.Improved muscle healing after contusion injury by the inhibitory effect of suramin on myostatin,a negative regulator of muscle growth[J].The American Journal of Sports Medicine,2008,36(12):2354-2362
[16] 刘晓光,赵淋淋,曾志刚,等.小鼠骨骼肌损伤修复过程中肌再生调节因子和血管再生因子表达规律研究 [J].中国康复医学杂志,2016,31(12):1294-1300
[17] NOVAK M L,KOH T J.Macrophage phenotypes during tissue repair[J].Journal of Leukocyte Biology,2013,93(6):875-881
[18] PILLON N J,BILAN P J,FINK L N,et al.Cross-talk between skeletal muscle and immune cells:Muscle-derived mediators and metabolic implications[J].American Journal of Physiology Endocrinology and Metabolism,2013,304(5):E453-465
[19] KIMURA N,HIRATA S,MIYASAKA N,et al.Injury and subsequent regeneration of muscles for activation of local innate immunity to facilitate the development and relapse of autoimmune myositis in C57BL/6 mice [J].Arthritis & Rheumatology,2015,67(4):1107-1116
[20] TONKIN J,TEMMERMAN L,SAMPSON R D,et al.Monocyte/macrophage-derived IGF-1 orchestrates murine skeletal muscle regeneration and modulates autocrine polarization[J].Molecular Therapy:The Journal of The American Society of Gene Therapy,2015,23(7):1189-1200
[21] SHONO J,SAKAGUCHI S,SUZUKI T,et al.Preliminary time-course study of antiinflammatory macrophage infiltration in crush-injured skeletal muscle[J].Animal Science Journal=Nihon Chikusan Gakkaiho,2013,84(11):744-750
[22] DENG B,WEHLING H M,VILLALTA S A,et al.IL-10 triggers changes in macrophage phenotype that promote muscle growth and regeneration [J].Journal of Immunology,2012,189(7):3669-3680
[23] ZOU K,WANG Y,HU Y,et al.Specific tumor-derived CCL2 mediated by pyruvate kinase M2 in colorectal cancer cells contributes to macrophage recruitment in tumor microenvironment[J].Tumour Biol,2017,39(3):1-10
[24] 杨建斌,冯红超,宋宇峰.口腔鳞癌中单核细胞趋化蛋白-1在巨噬细胞浸润、聚集中的意义 [J].中国肿瘤临床,2005,32(20):1155-1157
[25] CARSON W F T,SALTER G S E,SCOLA M M,et al.Enhancement of macrophage inflammatory responses by CCL2 is correlated with increased miR-9 expression and downregulation of the ERK1/2 phosphatase Dusp6 [J].Cell Immunol,2017,314:63-72
[26] CONTRERAS S V,OCHOA O,REYES R S M,et al.Fat accumulation with altered inflammation and regeneration in skeletal muscle of CCR2-/- mice following ischemic injury[J].American Journal of Physiology Cell Physiology,2007,292(2):C953-967
[27] OCHOA O,SUN D,REYES R S M,et al.Delayed angiogenesis and VEGF production in CCR2-/- mice during impaired skeletal muscle regeneration [J].American Journal of Physiology Regulatory,Integrative and Comparative Physiology,2007,293(2):R651-661
[28] ARNOLD L,HENRY A,PORON F,et al.Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory macrophages to support myogenesis [J].J Exp Med,2007,204(5):1057-1069
[29] BRZOSKA E,KOWALSKI K,MARKOWSKA Z A,et al.Sdf-1 (CXCL12) induces CD9 expression in stem cells engaged in muscle regeneration[J].Stem Cell Research & Therapy,2015,6:46
[30] BRZOSKA E,KOWALEWSKA M,MARKOWSKA Z A,et al.Sdf-1 (CXCL12) improves skeletal muscle regeneration via the mobilisation of Cxcr4 and CD34 expressing cells[J].Biology of The Cell/Under The Auspices of The European Cell Biology Organization,2012,104(12):722-737
[31] RATAJCZAK M Z,MAJKA M,KUCIA M,et al.Expression of functional CXCR4 by muscle satellite cells and secretion of SDF-1 by muscle-derived fibroblasts is associated with the presence of both muscle progenitors in bone marrow and hematopoietic stem/progenitor cells in muscles[J].Stem Cells,2003,21(3):363-371
[32] 杨志峰,杨清玲,陈昌杰.趋化因子SDF-1与受体CXCR4的研究进展 [J].分子诊断与治疗杂志,2011,3(1):58-61
[33] 李鹏程,徐茜,胡燕华.蛋白激酶C对SDF-1诱导巨噬细胞趋化及血管内皮生长因子转录的调节作用 [J].华中科技大学学报(医学版),2008,37(1):93-96