脂多糖刺激下骨髓间充质干细胞分泌的外泌体对Ly6C单核巨噬细胞亚群的影响
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摘要
目的探索炎症刺激下,骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)产生的外泌体对小鼠淋巴抗原6C高表达(Ly6C~(high))单核巨噬细胞和Ly6C~(low)两个亚群比例的影响。方法梯度离心法分离BMSCs,鉴定其形态、表面标记及成脂诱导分化能力。脂多糖处理BMSCs 48h,收集上清并提取外泌体,采用Western blot法、透射电镜以及粒径分析鉴定外泌体。磁珠分选小鼠腿骨中的单核巨噬细胞,与外泌体共培养24 h后,LPS刺激48 h,流式细胞术检测Ly6C亚群变化。ELISA检测细胞上清炎症因子的变化。结果 BMSCs表达CD44、CD90等,不表达CD34、CD45,并有成脂分化能力。外泌体表达CD63,粒径大小为(82.4±3.70)nm,LPS刺激下BMSCs产生较多的外泌体(P<0.01)。与LPS组相比,外泌体可以使Ly6C~(high)单核细胞比例上升(P<0.01),Ly6C~(high)巨噬细胞比例下降(P<0.05),且LPS刺激BMSCs产生的外泌体比正常BMSCs产生的外泌体的作用更加明显(P<0.05)。促炎因子IL-6水平也明显上调(P<0.05)。结论 BMSCs产生的外泌体可以改变Ly6C~(high)单核巨噬细胞亚群比例,为BMSCs的临床治疗提供了新的靶点。
Aim To determine the effect of exosomes from lipopolysaccharide-treated human bone marrow mesenchymal stem cells on proportion of Ly6C~(high) and Ly6C~(low) monocytes/macrophages in inflammatory microenvironment.Methods BMSCs were obtained by gradient centrifugation,identified and then treated with lipopolysaccharide for 48 h.The exosomes were purified from conditional medium with or without LPS treatment and identified by CD63 protein using Western blot and transmission electron microscope.The diameters and concentration were detected by Nanoparticle Trafficking Analysis( NTA).The monocytes/macrophages were sorted from bone marrow of the mice by magnetic beads.Cells were co-cultured with exosomes for 24 hours,and then treated with LPS for 48 hours.The proportion of Ly6C monocytes/macrophages was detected by flow cytometry.Inflammatory cytokines in cell supernatant were investigated using ELISA.Results BMSCs surface markers CD44,CD90 were positively detected,but CD34,CD45 were not expressed.BMSCs presented adipogenic differentiation ability.Exosomes were positively expressing CD63 protein,and NTA showed that the diameters of exosomes were up to( 82.4 ± 3.7) nm.BMSCs stimulated by LPS produced more exosomes( P < 0.01).Exosomes from BMSCs with or without LPS treatment could increase the ratio of Ly6C~(high) monocytes( P < 0.01) and downregulate the ratio of Ly6C~(high) macrophages( P < 0.05),and the effect of LPS treated-exosomes was more significant than untreated-exosomes( P < 0.05).Moreover,the concentration of IL-6 was also elevated under exosomes treatment( P < 0.05).Conclusions Human bone marrow mesenchymal stem cells-derived exosomes contribute to the regulation of Ly6C~(high) monocytes/macrophages,indicating that they could be involved in the therapeutic treatment of inflammatory diseases.
Aim To determine the effect of exosomes from lipopolysaccharide-treated human bone marrow mesenchymal stem cells on proportion of Ly6C~(high) and Ly6C~(low) monocytes/macrophages in inflammatory microenvironment.Methods BMSCs were obtained by gradient centrifugation,identified and then treated with lipopolysaccharide for 48 h.The exosomes were purified from conditional medium with or without LPS treatment and identified by CD63 protein using Western blot and transmission electron microscope.The diameters and concentration were detected by Nanoparticle Trafficking Analysis( NTA).The monocytes/macrophages were sorted from bone marrow of the mice by magnetic beads.Cells were co-cultured with exosomes for 24 hours,and then treated with LPS for 48 hours.The proportion of Ly6C monocytes/macrophages was detected by flow cytometry.Inflammatory cytokines in cell supernatant were investigated using ELISA.Results BMSCs surface markers CD44,CD90 were positively detected,but CD34,CD45 were not expressed.BMSCs presented adipogenic differentiation ability.Exosomes were positively expressing CD63 protein,and NTA showed that the diameters of exosomes were up to( 82.4 ± 3.7) nm.BMSCs stimulated by LPS produced more exosomes( P < 0.01).Exosomes from BMSCs with or without LPS treatment could increase the ratio of Ly6C~(high) monocytes( P < 0.01) and downregulate the ratio of Ly6C~(high) macrophages( P < 0.05),and the effect of LPS treated-exosomes was more significant than untreated-exosomes( P < 0.05).Moreover,the concentration of IL-6 was also elevated under exosomes treatment( P < 0.05).Conclusions Human bone marrow mesenchymal stem cells-derived exosomes contribute to the regulation of Ly6C~(high) monocytes/macrophages,indicating that they could be involved in the therapeutic treatment of inflammatory diseases.
引文
[1]Das A,Sinha M,Datta S,et al.Monocyte and macrophage plasticity in tissue repair and regeneration[J].Am J Pathol,2015,185(10):2596-606.
[2]Hilgendorf I,Gerhardt L M,Tan T C,et al.Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium[J].Circ Res,2014,114(10):1611-22.
[3]Lin S L,Casta1o A P,Nowlin B T,et al.Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations[J].J Immunol,2009,183(10):6733-43.
[4]Dayan V,Yannarelli G,Billia F,et al.Mesenchymal stromal cells mediate a switch to alternatively activated monocytes/macrophages after acute myocardial infarction[J].Basic Res Cardiol,2011,106(6):1299-310.
[5]江新湄,林秋雄,何国东,等.青钱柳降糖组方对2型糖尿病大鼠的治疗作用[J].中国病理生理杂志,2017,33(10):1794-1800.[5]Jiang X M,Lin Q X,He G D,et al.Therapeutic effect of QQL prescription on type 2 diabetic rats[J].Chin J Pathol,2017,33(10):1794-1800.
[6]Ibrahim A,Marbán E.Exosomes:fundamental biology and roles in cardiovascular physiology[J].Annu Rev Physiol,2016,78:67-83.
[7]Nahrendorf M,Swirski F K,Aikawa E,et al.The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions[J].J Exp Med,2007,204(12):3037-47.
[8]Lombardo E,van der Poll T,de la Rosa O,et al.Mesenchymal stem cells as a therapeutic tool to treat sepsis[J].World J Stem Cells,2015,7(2):368.
[9]Yao Y,Zhang F,Wang L,et al.Lipopolysaccharide preconditioning enhances the efficacy of mesenchymal stem cells transplantation in a rat model of acute myocardial infarction[J].J Biomed Sci,2009,16:74.
[10]杨翔宇,李晓红,肖静,等.干扰素γ刺激h UC-MSCs分泌外泌体促进调节性T细胞生成[J].中国药理学通报,2017,33(1):45-51.[10]Yang X Y,Li X H,Xiao J,et al.Exosomes secreted from IFN-γprestimulated h UC-MSCs induce regulatory T cells[J].Chin Pharmacol Bull,2017,33(1):45-51.
[11]李超,戴敏.不同细胞来源的外泌体在动脉粥样硬化中的研究进展[J].中国药理学通报,2017,33(1):27-32.[11]Li C,Dai M.Research progress of different cells derived exosomes in atherosclerosis[J].Chin Pharmacol Bull,2017,33(1):27-32.
[12]肖静,潘宇,李晓红,等.心脏干细胞分泌的exosome对氧化应激下心肌细胞的保护作用研究[J].中国药理学通报,2015,31(12):1656-60.[12]Xiao J,Pan Y,Li X H,et al.CPC derived exosome protects cardiomyocytes from oxidative stress[J].Chin Pharmacol Bull,2015,31(12):1656-60.
[13]Kilroy G E,Foster S J,Wu X,et al.Cytokine profile of human adipose‐derived stem cells:expression of angiogenic,hematopoietic,and pro-inflammatory factors[J].J Cell Physiol,2007,212(3):702-9.
[14]Lee S C,Jeong H J,Lee S K,et al.Lipopolysaccharide preconditioning of adipose-derived stem cells improves liver-regenerating activity of the secretome[J].Stem Cell Res Ther,2015,6(1):75.
[15]Bunnell B A,Betancourt A M,Sullivan D E.New concepts on the immune modulation mediated by mesenchymal stem cells[J].Stem Cell Res Ther,2010,1(5):34.
[2]Hilgendorf I,Gerhardt L M,Tan T C,et al.Ly-6Chigh monocytes depend on Nr4a1 to balance both inflammatory and reparative phases in the infarcted myocardium[J].Circ Res,2014,114(10):1611-22.
[3]Lin S L,Casta1o A P,Nowlin B T,et al.Bone marrow Ly6Chigh monocytes are selectively recruited to injured kidney and differentiate into functionally distinct populations[J].J Immunol,2009,183(10):6733-43.
[4]Dayan V,Yannarelli G,Billia F,et al.Mesenchymal stromal cells mediate a switch to alternatively activated monocytes/macrophages after acute myocardial infarction[J].Basic Res Cardiol,2011,106(6):1299-310.
[5]江新湄,林秋雄,何国东,等.青钱柳降糖组方对2型糖尿病大鼠的治疗作用[J].中国病理生理杂志,2017,33(10):1794-1800.[5]Jiang X M,Lin Q X,He G D,et al.Therapeutic effect of QQL prescription on type 2 diabetic rats[J].Chin J Pathol,2017,33(10):1794-1800.
[6]Ibrahim A,Marbán E.Exosomes:fundamental biology and roles in cardiovascular physiology[J].Annu Rev Physiol,2016,78:67-83.
[7]Nahrendorf M,Swirski F K,Aikawa E,et al.The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions[J].J Exp Med,2007,204(12):3037-47.
[8]Lombardo E,van der Poll T,de la Rosa O,et al.Mesenchymal stem cells as a therapeutic tool to treat sepsis[J].World J Stem Cells,2015,7(2):368.
[9]Yao Y,Zhang F,Wang L,et al.Lipopolysaccharide preconditioning enhances the efficacy of mesenchymal stem cells transplantation in a rat model of acute myocardial infarction[J].J Biomed Sci,2009,16:74.
[10]杨翔宇,李晓红,肖静,等.干扰素γ刺激h UC-MSCs分泌外泌体促进调节性T细胞生成[J].中国药理学通报,2017,33(1):45-51.[10]Yang X Y,Li X H,Xiao J,et al.Exosomes secreted from IFN-γprestimulated h UC-MSCs induce regulatory T cells[J].Chin Pharmacol Bull,2017,33(1):45-51.
[11]李超,戴敏.不同细胞来源的外泌体在动脉粥样硬化中的研究进展[J].中国药理学通报,2017,33(1):27-32.[11]Li C,Dai M.Research progress of different cells derived exosomes in atherosclerosis[J].Chin Pharmacol Bull,2017,33(1):27-32.
[12]肖静,潘宇,李晓红,等.心脏干细胞分泌的exosome对氧化应激下心肌细胞的保护作用研究[J].中国药理学通报,2015,31(12):1656-60.[12]Xiao J,Pan Y,Li X H,et al.CPC derived exosome protects cardiomyocytes from oxidative stress[J].Chin Pharmacol Bull,2015,31(12):1656-60.
[13]Kilroy G E,Foster S J,Wu X,et al.Cytokine profile of human adipose‐derived stem cells:expression of angiogenic,hematopoietic,and pro-inflammatory factors[J].J Cell Physiol,2007,212(3):702-9.
[14]Lee S C,Jeong H J,Lee S K,et al.Lipopolysaccharide preconditioning of adipose-derived stem cells improves liver-regenerating activity of the secretome[J].Stem Cell Res Ther,2015,6(1):75.
[15]Bunnell B A,Betancourt A M,Sullivan D E.New concepts on the immune modulation mediated by mesenchymal stem cells[J].Stem Cell Res Ther,2010,1(5):34.