脂肪干细胞来源外泌体对人脐静脉血管内皮细胞增殖、迁移及管样分化的影响
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摘要
目的探讨脂肪干细胞来源外泌体(adipose-derived stem cell released exosomes,ADSC-Exos)对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖、迁移及管样分化的影响。方法取吸脂患者自愿捐赠的脂肪组织,采用酶消化法分离培养获得ADSCs,并行流式细胞检测及成脂诱导鉴定。收集第3代ADSCs上清液提取外泌体,透射电镜观察其形态,Western blot检测其膜表面标志性蛋白Alix和CD63,用纳米颗粒跟踪分析仪NanoSight分析外泌体粒径分布范围。用PKH26荧光标记的ADSC-Exos与HUVECs共培养后,共聚焦显微镜观察ADSC-Exos能否进入HUVECs。将ADSC-Exos与HUVECs共培养1、2、3、4、5 d,CCK-8法检测ADSC-Exos对HUVECs增殖影响;共培养12 h时ELISA法检测细胞上清液VEGF蛋白表达量。采用Transwell迁移实验检测ADSC-Exos对HUVECs迁移能力的影响。通过体外Matrigel基质胶实验,观察ADSCExos对HUVECs管状结构形成的影响;将HUVECs与Matrigel基质胶混合后联合ADSC-Exos注射在BALB/c雄性裸鼠皮下,2周后检测基质胶中新生血管情况,计算平均血管密度(mean blood vessel density,MVD)。上述实验均以等量PBS作为对照。结果经鉴定所培养细胞符合ADSCs的特征。ADSC-Exos为形态一致的圆形或椭圆形膜性囊泡,表达标志性蛋白Alix和CD63,粒径范围30~200 nm。共聚焦显微镜观察示ADSC-Exos可被HUVECs摄取。CCK-8法检测示,各时间点实验组细胞增殖均优于对照组(P<0.05)。Transwell实验结果显示,实验组跨膜迁移细胞数较对照组显著增多(t=9.534,P=0.000)。在体外Matrigel胶成管实验中,实验组管样结构数明显多于对照组(t=15.910,P=0.000);在体内实验中,实验组MVD亦显著高于对照组(t=16.710,P=0.000)。ELISA检测示,实验组细胞上清液VEGF蛋白表达量明显高于对照组(t=21.470,P=0.000)。结论 ADSC-Exos可促进HUVECs增殖、迁移及管样结构形成,提示ADSC-Exos可促进血管新生。
Objective To explore the effects of adipose-derived stem cell released exosomes(ADSC-Exos)on the proliferation, migration, and tube-like differentiation of human umbilical vein endothelial cells(HUVECs).Methods Adipose tissue voluntarily donated by liposuction patients was obtained. The ADSCs were harvested by enzyme digestion and identified by flow cytometry and adipogenic induction. The ADSC-Exos were extracted from the supernatant of the 3 rd generation ADSCs and the morphology was observed by transmission electron microscopy. The surface proteins(Alix and CD63) were detected by Western blot. The nanoparticle tracking analyzer NanoSight was used to analyze the size distribution of ADSC-Exos. After co-culture of PKH26 fluorescently labeled ADSC-Exos with HUVECs, confocal microscopy had been used to observe whether ADSC-Exos could absorbed by HUVECs. ADSC-Exos and HUVECs were co-cultured for 1, 2, 3, 4, and 5 days. The effect of ADSC-Exos on the proliferation of HUVECs was detected by cell counting kit 8(CCK-8) assay. The expression of VEGF protein in the supernatant of HUVECs with or without ADSC-Exos had been detected by ELISA after 12 hours. Transwell migration assay was used to detect the effect of ADSC-Exos on the migration ability of HUVECs. The effect of ADSC-Exos on the tubular structure formation of HUVECs was observed by Matrigel experiments in vitro. The formation of subcutaneous tubular structure in vivo was observed in BALB/c male nude mice via the injection of HUVECs and Matrigel with or without ADSC-Exos. After2 weeks, the neovascularization in Matrigel was measured and mean blood vessel density(MVD) was calculated. The above experiments were all controlled by the same amount of PBS. Results After identification, the cultured cells were consistent with the characteristics of ADSCs. ADSC-Exos were circular or elliptical membranous vesicle with uniform morphology under transmission electron microscopy, and expresses the signature proteins Alix and CD63 with particle size ranging from 30 to 200 nm. Confocal microscopy results showed that ADSC-Exos could be absorbed by HUVECs.The CCK-8 analysis showed that the cell proliferation of the experimental group was better than that of the control group at each time point(P<0.05). The result of Transwell showed that the trans-membrane migration cells in the experimental group were significantly more than that in the control group(t=9.534, P=0.000). In vitro, Matrigel tube-forming experiment showed that the number of tube-like structures in the experimental group was significantly higher than that of the control group(t=15.910, P=0.000). In vivo, the MVD of the experimental group was significantly higher than that of the control group(t=16.710, P=0.000). The ELISA assay showed that the expression of VEGF protein in the supernatant of the experimental group was significantly higher than that of the control group(t=21.470, P=0.000). Conclusion ADSCExos can promote proliferation, migration, and tube-like structure formation of HUVECs, suggesting that ADSC-Exos can promote angiogenesis in vitro and in vivo.
Objective To explore the effects of adipose-derived stem cell released exosomes(ADSC-Exos)on the proliferation, migration, and tube-like differentiation of human umbilical vein endothelial cells(HUVECs).Methods Adipose tissue voluntarily donated by liposuction patients was obtained. The ADSCs were harvested by enzyme digestion and identified by flow cytometry and adipogenic induction. The ADSC-Exos were extracted from the supernatant of the 3 rd generation ADSCs and the morphology was observed by transmission electron microscopy. The surface proteins(Alix and CD63) were detected by Western blot. The nanoparticle tracking analyzer NanoSight was used to analyze the size distribution of ADSC-Exos. After co-culture of PKH26 fluorescently labeled ADSC-Exos with HUVECs, confocal microscopy had been used to observe whether ADSC-Exos could absorbed by HUVECs. ADSC-Exos and HUVECs were co-cultured for 1, 2, 3, 4, and 5 days. The effect of ADSC-Exos on the proliferation of HUVECs was detected by cell counting kit 8(CCK-8) assay. The expression of VEGF protein in the supernatant of HUVECs with or without ADSC-Exos had been detected by ELISA after 12 hours. Transwell migration assay was used to detect the effect of ADSC-Exos on the migration ability of HUVECs. The effect of ADSC-Exos on the tubular structure formation of HUVECs was observed by Matrigel experiments in vitro. The formation of subcutaneous tubular structure in vivo was observed in BALB/c male nude mice via the injection of HUVECs and Matrigel with or without ADSC-Exos. After2 weeks, the neovascularization in Matrigel was measured and mean blood vessel density(MVD) was calculated. The above experiments were all controlled by the same amount of PBS. Results After identification, the cultured cells were consistent with the characteristics of ADSCs. ADSC-Exos were circular or elliptical membranous vesicle with uniform morphology under transmission electron microscopy, and expresses the signature proteins Alix and CD63 with particle size ranging from 30 to 200 nm. Confocal microscopy results showed that ADSC-Exos could be absorbed by HUVECs.The CCK-8 analysis showed that the cell proliferation of the experimental group was better than that of the control group at each time point(P<0.05). The result of Transwell showed that the trans-membrane migration cells in the experimental group were significantly more than that in the control group(t=9.534, P=0.000). In vitro, Matrigel tube-forming experiment showed that the number of tube-like structures in the experimental group was significantly higher than that of the control group(t=15.910, P=0.000). In vivo, the MVD of the experimental group was significantly higher than that of the control group(t=16.710, P=0.000). The ELISA assay showed that the expression of VEGF protein in the supernatant of the experimental group was significantly higher than that of the control group(t=21.470, P=0.000). Conclusion ADSCExos can promote proliferation, migration, and tube-like structure formation of HUVECs, suggesting that ADSC-Exos can promote angiogenesis in vitro and in vivo.
引文
1Rao RR,Stegemann JP.Cell-based approaches to the engineering of vascularized bone tissue.Cytotherapy,2013,15(11):1309-1322.
2Rohringer S,Hofbauer P,Schneider KH,et al.Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells.Angiogenesis,2014,17(4):921-933.
3Than U,Guanzon D,Leavesley D,et al.Association of extracellular membrane vesicles with cutaneous wound healing.Int J Mol Sci,2017,18(5):E956.
4Franquesa M,Hoogduijn MJ,Ripoll E,et al.Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells.Front Immunol,2014,5:525.
5Riazifar M,Pone EJ,L?tvall J,et al.Stem cell extracellular vesicles:extended messages of regeneration.Annu Rev Pharmacol Toxicol,2017,57:125-154.
6Li Q,Shao Y,Zhang X,et al.Plasma long noncoding RNA protected by exosomes as a potential stable biomarker for gastric cancer.Tumour Biol,2015,36(3):2007-2012.
7Chen B,Li Q,Zhao B,et al.Stem cell-derived extracellular vesicles as a novel potential therapeutic tool for tissue repair.Stem Cells Transl Med,2017,6(9):1753-1758.
8Deveza L,Choi J,Imanbayev G,et al.Paracrine release from nonviral engineered adipose-derived stem cells promotes endothelial cell survival and migration in vitro.Stem Cells Dev,2013,22(3):483-491.
9Johnstone RM,Adam M,Hammond JR,et al.Vesicle formation during reticulocyte maturation.Association of plasma membrane activities with released vesicles(exosomes).J Biol Chem,1987,262(19):9412-9420.
10Cocucci E,Meldolesi J.Ectosomes and exosomes:shedding the confusion between extracellular vesicles.Trends Cell Biol,2015,25(6):364-372.
11Konala VB,Mamidi MK,Bhonde R,et al.The current landscape of the mesenchymal stromal cell secretome:A new paradigm for cell-free regeneration.Cytotherapy,2016,18(1):13-24.
12Bj?rge IM,Kim SY,Mano JF,et al.Extracellular vesicles,exosomes and shedding vesicles in regenerative medicine-a new paradigm for tissue repair.Biomater Sci,2017,6(1):60-78.
13Randi AM,Laffan MA.Von Willebrand factor and angiogenesis:basic and applied issues.J Thromb Haemost,2017,15(1):13-20.
14Eelen G,de Zeeuw P,Simons M,et al.Endothelial cell metabolism in normal and diseased vasculature.Circ Res,2015,116(7):1231-1244.
15Logsdon EA,Finley SD,Popel AS,et al.A systems biology view of blood vessel growth and remodeling.J Cell Mol Med,2014,18(8):1491-1508.
16Hessvik NP,Llorente A.Current knowledge on exosome biogenesis and release.Cell Mol Life Sci,2018,75(2):193-208.
17Webber J,Clayton A.How pure are your vesicles?J Extracell Vesicles,2013,2:10.
18Kishore R,Khan M.More Than Tiny Sacks:Stem cell exosomes as cell-free modality for cardiac repair.Circ Res,2016,118(2):330-343.
19Ozturk S,Elcin YM.Cardiac stem cell characteristics in physiological and pathological conditions.Curr Pharm Des,2018.[Epub ahead of print]
20Liang X,Zhang L,Wang S,et al.Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a.J Cell Sci,2016,129(11):2182-2189.
2Rohringer S,Hofbauer P,Schneider KH,et al.Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells.Angiogenesis,2014,17(4):921-933.
3Than U,Guanzon D,Leavesley D,et al.Association of extracellular membrane vesicles with cutaneous wound healing.Int J Mol Sci,2017,18(5):E956.
4Franquesa M,Hoogduijn MJ,Ripoll E,et al.Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells.Front Immunol,2014,5:525.
5Riazifar M,Pone EJ,L?tvall J,et al.Stem cell extracellular vesicles:extended messages of regeneration.Annu Rev Pharmacol Toxicol,2017,57:125-154.
6Li Q,Shao Y,Zhang X,et al.Plasma long noncoding RNA protected by exosomes as a potential stable biomarker for gastric cancer.Tumour Biol,2015,36(3):2007-2012.
7Chen B,Li Q,Zhao B,et al.Stem cell-derived extracellular vesicles as a novel potential therapeutic tool for tissue repair.Stem Cells Transl Med,2017,6(9):1753-1758.
8Deveza L,Choi J,Imanbayev G,et al.Paracrine release from nonviral engineered adipose-derived stem cells promotes endothelial cell survival and migration in vitro.Stem Cells Dev,2013,22(3):483-491.
9Johnstone RM,Adam M,Hammond JR,et al.Vesicle formation during reticulocyte maturation.Association of plasma membrane activities with released vesicles(exosomes).J Biol Chem,1987,262(19):9412-9420.
10Cocucci E,Meldolesi J.Ectosomes and exosomes:shedding the confusion between extracellular vesicles.Trends Cell Biol,2015,25(6):364-372.
11Konala VB,Mamidi MK,Bhonde R,et al.The current landscape of the mesenchymal stromal cell secretome:A new paradigm for cell-free regeneration.Cytotherapy,2016,18(1):13-24.
12Bj?rge IM,Kim SY,Mano JF,et al.Extracellular vesicles,exosomes and shedding vesicles in regenerative medicine-a new paradigm for tissue repair.Biomater Sci,2017,6(1):60-78.
13Randi AM,Laffan MA.Von Willebrand factor and angiogenesis:basic and applied issues.J Thromb Haemost,2017,15(1):13-20.
14Eelen G,de Zeeuw P,Simons M,et al.Endothelial cell metabolism in normal and diseased vasculature.Circ Res,2015,116(7):1231-1244.
15Logsdon EA,Finley SD,Popel AS,et al.A systems biology view of blood vessel growth and remodeling.J Cell Mol Med,2014,18(8):1491-1508.
16Hessvik NP,Llorente A.Current knowledge on exosome biogenesis and release.Cell Mol Life Sci,2018,75(2):193-208.
17Webber J,Clayton A.How pure are your vesicles?J Extracell Vesicles,2013,2:10.
18Kishore R,Khan M.More Than Tiny Sacks:Stem cell exosomes as cell-free modality for cardiac repair.Circ Res,2016,118(2):330-343.
19Ozturk S,Elcin YM.Cardiac stem cell characteristics in physiological and pathological conditions.Curr Pharm Des,2018.[Epub ahead of print]
20Liang X,Zhang L,Wang S,et al.Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a.J Cell Sci,2016,129(11):2182-2189.