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.
作者
张静
易阳艳
阳水发
朱元正
胡玄
ZHANG Jing;YI Yangyan;YANG Shuifa;ZHU Yuanzheng;HU Xuan(Department of Plastic Surgery,the Second Affiliated Hospital of Nanchang University,Nanchang Jiangxi,330006,P.R.China)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2018年第10期1351-1357,共7页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(81660326)
江西省自然科学基金资助项目(20171ACB20037)~~
关键词
脂肪干细胞
外泌体
血管新生
人脐静脉血管内皮细胞
Adipose-derived stem cells
exosomes
angiogenesis
human umbilical vein endothelial cells
