人脐带间充质干细胞来源外泌体可调控巨噬细胞的极化
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摘要
背景:间充质干细胞来源外泌体具有与母细胞类似的修复组织损伤的作用,但机制尚未完全阐明。巨噬细胞在组织损伤修复过程中扮演重要角色。因此,探讨间充质干细胞来源外泌体对巨噬细胞极化的调控作用,将有助于阐明其组织修复的机制。目的:提取人脐带间充质干细胞来源外泌体,并探讨其对巨噬细胞极化的影响。方法:采用超速离心法提取人脐带间充质干细胞分泌的外泌体,分别采用透射电镜观察其形态、纳米颗粒追踪法检测粒径、蛋白印迹法检测标志蛋白表达;然后用得到的外泌体处理小鼠骨髓巨噬细胞,探讨其对巨噬细胞极化的调控作用。结果与结论:①成功提取人脐带间充质干细胞来源外泌体,其形态呈经典的囊泡样结构,直径(92.0±34.1) nm,表达外泌体标志蛋白CD63和Alix;②该外泌体能显著降低脂多糖诱导的巨噬细胞M1型活化标志肿瘤坏死因子α和一氧化氮合酶水平,而提高M2型活化标志白细胞介素10和精氨酸酶1水平;③结果可见,人脐带间充质干细胞来源外泌体具有调控巨噬细胞向M2型极化的作用,进而发挥组织修复的功能。
BACKGROUND: Accumulating evidence has suggested that mesenchymal stem cells-derived exosomes have the similar tissue repair activity compared to the mother cells. However, the underlying mechanisms are not yet illuminated. Since macrophages play pivotal roles during tissue repair process, we investigated the regulatory effect of mesenchymal stem cells-derived exosomes on macrophages, aiming to better elucidate its mechanism involved in tissue repair.OBJECTIVE: To isolate exosomes from human umbilical cord-derived mesenchymal stem cells, and to evaluate their regulatory effect on macrophage polarization.METHODS: Exosomes were isolated from the culture supernatants of human umbilical cord-derived mesenchymal stem cells by the differential centrifugation method, and then characterized by their morphology, size distribution and protein markers using transmission electron microscopy, nanoparticle tracking analysis and western blot assay, respectively. Furthermore, their regulatory effects on M1/M2 macrophage polarization were evaluated in mouse bone marrow-derived macrophages.RESULTS AND CONCLUSION: Exosomes were successfully isolated from human umbilical cord-derived mesenchymal stem cell supernatants with a cup-shaped canonical morphology,(92.0±34.1) nm in diameter, and protein markers of CD63 and Alix. We determined that these exosomes could significantly reduce lipopolysaccharide-induced activation of M1 markers tumor necrosis factor-α and inducible nitric oxide, while upregulating M2 markers interleukin-10 andarginase-1. These findings suggest that exosomes from human umbilical cord-derived mesenchymal stem cells can regulate macrophage polarization to M2 phenotype, benefiting tissue repairing.
BACKGROUND: Accumulating evidence has suggested that mesenchymal stem cells-derived exosomes have the similar tissue repair activity compared to the mother cells. However, the underlying mechanisms are not yet illuminated. Since macrophages play pivotal roles during tissue repair process, we investigated the regulatory effect of mesenchymal stem cells-derived exosomes on macrophages, aiming to better elucidate its mechanism involved in tissue repair.OBJECTIVE: To isolate exosomes from human umbilical cord-derived mesenchymal stem cells, and to evaluate their regulatory effect on macrophage polarization.METHODS: Exosomes were isolated from the culture supernatants of human umbilical cord-derived mesenchymal stem cells by the differential centrifugation method, and then characterized by their morphology, size distribution and protein markers using transmission electron microscopy, nanoparticle tracking analysis and western blot assay, respectively. Furthermore, their regulatory effects on M1/M2 macrophage polarization were evaluated in mouse bone marrow-derived macrophages.RESULTS AND CONCLUSION: Exosomes were successfully isolated from human umbilical cord-derived mesenchymal stem cell supernatants with a cup-shaped canonical morphology,(92.0±34.1) nm in diameter, and protein markers of CD63 and Alix. We determined that these exosomes could significantly reduce lipopolysaccharide-induced activation of M1 markers tumor necrosis factor-α and inducible nitric oxide, while upregulating M2 markers interleukin-10 andarginase-1. These findings suggest that exosomes from human umbilical cord-derived mesenchymal stem cells can regulate macrophage polarization to M2 phenotype, benefiting tissue repairing.
引文
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[34]Maggini J,Mirkin G,Bognanni I,et al.Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.PLoS One.2010;5(2):e9252.
[35]Zhang QZ,Su WR,Shi SH,et al.Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing.Stem Cells.2010;28(10):1856-1868.
[36]Dayan V,Yannarelli G,Billia F,et al.Mesenchymal stromal cells mediate a switch to alternatively activated monocytes/macrophages after acute myocardial infarction.Basic Res Cardiol.2011;106(6):1299-1310.
[37]Guilloton F,Caron G,Ménard C,et al.Mesenchymal stromal cells orchestrate follicular lymphoma cell niche through the CCL2-dependent recruitment and polarization of monocytes.Blood.2012;119(11):2556-2567.
[38]Yin Y,Hao H,Cheng Y,et al.Human umbilical cord-derived mesenchymal stem cells direct macrophage polarization to alleviate pancreatic islets dysfunction in type 2 diabetic mice.Cell Death Dis.2018;9(7):760.
[39]Lankford KL,Arroyo EJ,Nazimek K,et al.Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord.PLoS One.2018;13(1):e0190358.
[40]Willis GR,Fernandez-Gonzalez A,Anastas J,et al.Mesenchymal Stromal Cell Exosomes Ameliorate Experimental Bronchopulmonary Dysplasia and Restore Lung Function through Macrophage Immunomodulation.Am J Respir Crit Care Med.2018;197(1):104-116.
[2]Le Blanc K,Tammik C,Rosendahl K,et al.HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells.Exp Hematol.2003;31(10):890-896.
[3]Le Blanc K,Tammik L,Sundberg B,et al.Mesenchymal stem cells inhibit and stimulate mixed lymphocyte cultures and mitogenic responses independently of the major histocompatibility complex.Scand J Immunol.2003;57(1):11-20.
[4]Rosario CM,Yandava BD,Kosaras B,et al.Differentiation of engrafted multipotent neural progenitors towards replacement of missing granule neurons in meander tail cerebellum may help determine the locus of mutant gene action.Development.1997;124(21):4213-4224.
[5]Li TS,Takahashi M,Ohshima M,et al.Myocardial repair achieved by the intramyocardial implantation of adult cardiomyocytes in combination with bone marrow cells.Cell Transplant.2008;17(6):695-703.
[6]Lai RC,Chen TS,Lim SK.Mesenchymal stem cell exosome:a novel stem cell-based therapy for cardiovascular disease.Regen Med.2011;6(4):481-492.
[7]Shi H,Xu X,Zhang B,et al.3,3'-Diindolylmethane stimulates exosomal Wnt11 autocrine signaling in human umbilical cord mesenchymal stem cells to enhance wound healing.Theranostics.2017;7(6):1674-1688.
[8]Vizoso FJ,Eiro N,Cid S,et al.Mesenchymal Stem Cell Secretome:Toward Cell-Free Therapeutic Strategies in Regenerative Medicine.Int J Mol Sci.2017;18(9):E1852.
[9]Vlassov AV,Magdaleno S,Setterquist R,et al.Exosomes:current knowledge of their composition,biological functions,and diagnostic and therapeutic potentials.Biochim Biophys Acta.2012;1820(7):940-948.
[10]Barile L,Vassalli G.Exosomes:Therapy delivery tools and biomarkers of diseases.Pharmacol Ther.2017;174:63-78.
[11]Huang L,Ma W,Ma Y,et al.Exosomes in mesenchymal stem cells,a new therapeutic strategy for cardiovascular diseases.Int J Biol Sci.2015;11(2):238-245.
[12]Zhang B,Wang M,Gong A,et al.HucMSC-Exosome MediatedWnt4 Signaling Is Required for Cutaneous Wound Healing.Stem Cells.2015;33(7):2158-2168.
[13]Zhang S,Chuah SJ,Lai RC,et al.MSC exosomes mediate cartilage repair by enhancing proliferation,attenuating apoptosis and modulating immune reactivity.Biomaterials.2018;156:16-27.
[14]Keshtkar S,Azarpira N,Ghahremani MH.Mesenchymal stem cell-derived extracellular vesicles:novel frontiers in regenerative medicine.Stem Cell Res Ther.2018;9(1):63.
[15]Gordon S,Taylor PR.Monocyte and macrophage heterogeneity.Nat Rev Immunol.2005;5(12):953-964.
[16]Sica A,Mantovani A.Macrophage plasticity and polarization:in vivo veritas.J Clin Invest.2012;122(3):787-795.
[17]Mantovani A,Sica A,Sozzani S,et al.The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol.2004;25(12):677-686.
[18]Martinez FO.Regulators of macrophage activation.Eur J Immunol.2011;41(6):1531-1534.
[19]Théry C,Amigorena S,Raposo G,et al.Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr Protoc Cell Biol.2006;Chapter 3:Unit 3.22.
[20]Lv FJ,Tuan RS,Cheung KM,et al.Concise review:the surface markers and identity of human mesenchymal stem cells.Stem Cells.2014;32(6):1408-1419.
[21]Zaborowski MP,Balaj L,Breakefield XO,et al.Extracellular Vesicles:Composition,Biological Relevance,and Methods of Study.Bioscience.2015;65(8):783-797.
[22]Théry C,Ostrowski M,Segura E.Membrane vesicles as conveyors of immune responses.Nat Rev Immunol.2009;9(8):581-593.
[23]Tolar J,Le Blanc K,Keating A,et al.Concise review:hitting the right spot with mesenchymal stromal cells.Stem Cells.2010;28(8):1446-1455.
[24]Kidd S,Spaeth E,Watson K,et al.Origins of the tumor microenvironment:quantitative assessment of adipose-derived and bone marrow-derived stroma.PLoS One.2012;7(2):e30563.
[25]Li J,Liu K,Liu Y,et al.Exosomes mediate the cel-to-cel transmission of IFN-α-induced antiviral activity.Nat Immunol.2013;14(8):793-803.
[26]Valadi H,Ekstr?m K,Bossios A,et al.Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol.2007;9(6):654-659.
[27]Ratajczak J,Miekus K,Kucia M,et al.Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors:evidence for horizontal transfer of mRNA and protein delivery.Leukemia.2006;20(5):847-856.
[28]Tkach M,Théry C.Communication by Extracellular Vesicles:Where We Are and Where We Need to Go.Cell.2016;164(6):1226-1232.
[29]Lai RC,Yeo RW,Lim SK.Mesenchymal stem cell exosomes.Semin Cell Dev Biol.2015;40:82-88.
[30]张恩国,陈尚雅,杨叶,等.干细胞源外泌体应用于再生医学的研究进展[J].中国组织工程研究,2018,22(5):801-806.
[31]Zhang B,Yeo RW,Tan KH,et al.Focus on Extracellular Vesicles:Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles.Int J Mol Sci.2016;17(2):174.
[32]Németh K,Leelahavanichkul A,Yuen PS,et al.Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production.Nat Med.2009;15(1):42-49.
[33]Kim J,Hematti P.Mesenchymal stem cell-educated macrophages:a novel type of alternatively activated macrophages.Exp Hematol.2009;37(12):1445-1453.
[34]Maggini J,Mirkin G,Bognanni I,et al.Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile.PLoS One.2010;5(2):e9252.
[35]Zhang QZ,Su WR,Shi SH,et al.Human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing.Stem Cells.2010;28(10):1856-1868.
[36]Dayan V,Yannarelli G,Billia F,et al.Mesenchymal stromal cells mediate a switch to alternatively activated monocytes/macrophages after acute myocardial infarction.Basic Res Cardiol.2011;106(6):1299-1310.
[37]Guilloton F,Caron G,Ménard C,et al.Mesenchymal stromal cells orchestrate follicular lymphoma cell niche through the CCL2-dependent recruitment and polarization of monocytes.Blood.2012;119(11):2556-2567.
[38]Yin Y,Hao H,Cheng Y,et al.Human umbilical cord-derived mesenchymal stem cells direct macrophage polarization to alleviate pancreatic islets dysfunction in type 2 diabetic mice.Cell Death Dis.2018;9(7):760.
[39]Lankford KL,Arroyo EJ,Nazimek K,et al.Intravenously delivered mesenchymal stem cell-derived exosomes target M2-type macrophages in the injured spinal cord.PLoS One.2018;13(1):e0190358.
[40]Willis GR,Fernandez-Gonzalez A,Anastas J,et al.Mesenchymal Stromal Cell Exosomes Ameliorate Experimental Bronchopulmonary Dysplasia and Restore Lung Function through Macrophage Immunomodulation.Am J Respir Crit Care Med.2018;197(1):104-116.
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