人脐带干细胞来源的外泌体促进体外培养雪旺细胞迁移的研究
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摘要
目的:外泌体是活细胞分泌的来源于多囊泡体的膜性囊泡,其主要作用包括携带与运输。雪旺细胞是周围神经再生中非常优秀的种子细胞,但其迁移能力较差,影响修复效果。本文旨在探讨外泌体和雪旺细胞共培养是否可以促进雪旺细胞迁移。方法:本实验通过分离纯化人脐带干细胞外泌体和大鼠坐骨神经雪旺细胞并鉴定,随后将其共培养于Transwell小室观察雪旺细胞迁移率。结果:通过人脐带干细胞超高速离心法得到的外泌体高表达干细胞标志物CD44(92.2±3.6%)、CD73(99.1±0.6%),并且低表达单核细胞表面抗原CD14(0.5±0.06%)以及造血干细胞表面抗原CD34(0.4±0.07%),外泌体鉴定高表达CD81和CD9;雪旺细胞培养鉴定纯度达(92.3±2.7)%;均符合实验要求。通过Transwell小室实验发现外泌体可以明显促进雪旺细胞的迁移,并且具有一定剂量关系。结论:外泌体可以提高雪旺细胞的迁移能力,从而使雪旺细胞在组织工程领域中的应用产生巨大突破。
Objective: The exosomes are membranous vesicles?which are derived from polycystic vesicles secreted by living cells.Their main functions include carrying and transporting substances. Schwann cells are one of the excellent seed cells in peripheral nerve regeneration. However, Schwann cells have a relatively poor migration ability which limite the biological effect on nerve regeneration.To investigate whether exosomes can promote Schwann cell migration under cell co-culture. Methods: In this experiment, we isolated and purified human umbilical cord stem cells' exosomes and Schwann cells from rat sciatic nerve and then identified them.Subsequently, they were co-cultured in Transwell chamber to observe the migration rate of Schwann cells. Results: High expression of stem cell markers CD44(92.2 ± 3.6 %), CD73(99.1 ± 0.6 %) and low expression of mononuclear cell surface antigen CD14(0.5 ± 0.06%), hematopoietic stem cell surface antigen CD34(0.4 ± 0.07 %) in h UC-MSCs which were obtained by ultrahigh speed centrifugation.The exosomes were identified with high expression of CD81 and CD9. The identification of Schwann cells were factual and the purity of cells were 92.3 ± 2.7 %. All the cultured cells and exosomes were met the experimental requirements. And the exosomes can significantly promote Schwann cells' migration under cell co-culture in Transwell chambers with dose relationship. Conclusions: The exosomes can accelerate Schwann cell migration obviously, which made Schwann cells more suitable for seed cells in nerve regeneration tissue engineering.
Objective: The exosomes are membranous vesicles?which are derived from polycystic vesicles secreted by living cells.Their main functions include carrying and transporting substances. Schwann cells are one of the excellent seed cells in peripheral nerve regeneration. However, Schwann cells have a relatively poor migration ability which limite the biological effect on nerve regeneration.To investigate whether exosomes can promote Schwann cell migration under cell co-culture. Methods: In this experiment, we isolated and purified human umbilical cord stem cells' exosomes and Schwann cells from rat sciatic nerve and then identified them.Subsequently, they were co-cultured in Transwell chamber to observe the migration rate of Schwann cells. Results: High expression of stem cell markers CD44(92.2 ± 3.6 %), CD73(99.1 ± 0.6 %) and low expression of mononuclear cell surface antigen CD14(0.5 ± 0.06%), hematopoietic stem cell surface antigen CD34(0.4 ± 0.07 %) in h UC-MSCs which were obtained by ultrahigh speed centrifugation.The exosomes were identified with high expression of CD81 and CD9. The identification of Schwann cells were factual and the purity of cells were 92.3 ± 2.7 %. All the cultured cells and exosomes were met the experimental requirements. And the exosomes can significantly promote Schwann cells' migration under cell co-culture in Transwell chambers with dose relationship. Conclusions: The exosomes can accelerate Schwann cell migration obviously, which made Schwann cells more suitable for seed cells in nerve regeneration tissue engineering.
引文
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[2]Dong H,Chen H,Jiang J,et al.Highly Sensitive Electrochemical Detection of Tumor Exosomes Based on Aptamer Recognitioninduced Multi-DNA Release and Cyclic Enzymatic Amplification[J]Anal Chem,2018,90(7):4507-4513
[3]Parimon T,Brauer R,Schlesinger SY,et al.Syndecan-1 Controls Lung Tumorigenesis by Regulating miRNAs Packaged in Exosomes[J].Am J Pathol,2018,188(4):1094-1103
[4]Chen C,Zong S,Wang Z,et al.Visualization and intracellular dynamic tracking of exosomes and exosomal miRNAs using single molecule localization microscopy[J].Nanoscale,2018,10(11):5154-5162
[5]Lian Q,Xu J,Yan S,et al.Chemotherapy-induced intestina inflammatory responses are mediated by exosome secretion of double-strand DNA via AIM2 inflammasome activation[J].Cell Res2017,27(6):784-800
[6]Cosenza S,Toupet K,Maumus M,et al.Mesenchymal stem cellsderived exosomes are more immunosuppressive than microparticles in inflammatory arthritis[J].Theranostics,2018,8(5):1399-1410
[7]Kimura K,Hohjoh H,Fukuoka M,et al.Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis[J].Nat Commun,2018,9(1):17
[8]Vicencio JM,Yellon DM,Sivaraman V,et al.Plasma exosomes protect the myocardium from ischemia-reperfusion injury[J].J Am Coll Cardiol,2015,65(15):1525-1536
[9]Tan JL,Lau SN,Leaw B,et al.Amnion Epithelial Cell-Derived Exosomes Restrict Lung Injury and Enhance Endogenous Lung Repair[J].Stem Cells Transl Med,2018,7(2):180-196
[10]JJ-P,Jansen K,Gramsbergen A,et al.Transection of periphera nerves,bridging strategies and effect evaluation[J].Biomaterials2004,25:1583-1592
[11]Lohmeyer JA,Siemers F,Machens HG,et al.The clinical use of artificial nerve conduits for digital nerve repair:a prospective cohort study and literature review[J].Journal of reconstructive microsurgery2009,25:055-061
[12]Mligiliche NL,Tabata Y,Kitada M,et al.Poly lactic acid caprolactone copolymer tube with a denatured skeletal muscle segment inside as a guide for peripheral nerve regeneration:Amorphological and electrophysiological evaluation of the regenerated nerves[J].Anatomical science international,2003,78:156-161
[13]Hood LJ,Brashears S,Long GR,et al.Understanding subtle changes in auditory function with otoacoustic emissions[J].Proceedings of Meetings on Acoustics:Acoustical Society of America,2013,050052
[14]Valero-Cabre A,Tsironis K,Skouras E,et al.Superior muscle reinnervation after autologous nerve graft or poly-L-lactideepsilon-caprolactone(PLC)tube implantation in comparison to silicone tube repair[J].Journal of neuroscience research,2001,63:214-223
[15]Evans GR.Peripheral nerve injury:a review and approach to tissue engineered constructs[J].The Anatomical record,2001,263:396-404
[16]Lambert WS,Clark AF,Wordinger RG,et al.Efect of exogenous neurotrophins on Trk receptor phosphorylation,cell proliferation,and neurotrophin secretion by cells isolated from the human lamina cribrosa[J].Mol Vis,2004,19(10):5289-5296
[17]Chao MV.Neurotrophin and their receptors:a convergence point for many signaling pathways[J].Nature Rev Neurosci,2003,4:299-309
[18]Hammarberg H,Risling M,H?kfelt T,et al.Expression of insulin like growth factors and corresponding binding proteins(IGFBP 1-6)in rat spinal cord and peripheral nerve after axonal injuries[J].Journal of Comparative Neurology,1998,400:57-72
[19]H觟ke A.Mechanisms of Disease:what factors limit the success of peripheral nerve regeneration in humans?[J].Nature Clinical Practice Neurology,2006,2:448-454
[20]Stang F,Fansa H,Wolf G,et al.Collagen nerve conduits-assessment of biocompatibility and axonal regeneration[J].Bio-medical materials and engineering,2005,15:3-12
[21]Fansa H,Dodic T,Wolf G,et al.Tissue engineering of pe ripheral nerves:epineurial grafts with application of cultured Schwann cells[J].Microsurgery,2003,23:72-77
[22]Allodi I,Udina E,Navarro X.Specificity of peripheral nerve regeneration:interactions at the axon level[J].Progress in neurobiology,2012,98:16-37
[23]Jiang X,Lim SH,Mao HQ,et al.Current applications and future perspectives of artificial nerve conduits[J].Experimental neurology,2010,223:86-101
[24]Itoh S,Suzuki M,Yamaguchi I,et al.Development of a nerve scaffold using a tendon chitosan tube[J].Artificial organs,2003,27:1079-1088
[25]Wang HB,Mullins ME,Cregg JM,et al.Creation of highly aligned electrospun poly-L-lactic acid fibers for nerve regeneration applications[J].Journal of neural engineering,2009,6:016001
[26]Arino H,Brandt J,Dahlin LB.Implantation of Schwann cells in rat tendon autografts as a model for peripheral nerve repair:long term effects on functional recovery[J].Scandinavian Journal of Plastic and Reconstructive Surgery and Hand Surgery,2008,42:281-285
[27]Xie Z,Wang X,Liu X,et al.Adipose-Derived Exosomes Exert Proatherogenic Effects by Regulating Macrophage Foam Cell Formation and Polarization[J].J Am Heart Assoc,2018,7(5)
[28]Hu M,Guo G,Huang Q,et al.The harsh microenvironment in infarcted heart accelerates transplanted bone marrow mesenchyma stem cells injury:the role of injured cardiomyocytes-derived exosomes[J].Cell Death Dis,2018,9(3):357
[29]Z?ller M.Janus-Faced Myeloid-Derived Suppressor Cell Exosomes for the Good and the Bad in Cancer and Autoimmune Disease[J]Front Immunol,2018,9:137
[30]Chen T,Xie MY,Sun JJ,et al.Porcine milk-derived exosomes promote proliferation of intestinal epithelial cells[J].Sci Rep,2016,6:33862
[31]Yu G,Jung H,Kang YY,et al.Comparative evaluation of cell-and serum-derived exosomes to deliver immune stimulators to lymph nodes[J].Biomaterials,2018,162:71-81
[32]Gheinani AH,V觟geli M,Baumgartner U,et al.Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery[J].Sci Rep,2018,8(1):3945