Jing Zhang1, Yangyan Yi2, Shuifa Yang1, Yuanzheng Zhu1, Xuan Hu1. 1. Department of Plastic Surgery, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China. 2. Department of Plastic Surgery, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China.yyy0218@126.com.
Abstract
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 3rd 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. After 2 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: ADSC-Exos 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 3rd 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. After 2 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: ADSC-Exos can promote proliferation, migration, and tube-like structure formation of HUVECs, suggesting that ADSC-Exos can promote angiogenesis in vitro and in vivo.
Authors: Jiangwen Wang; Yangyan Yi; Yuanzheng Zhu; Zhaohui Wang; Shu Wu; Jing Zhang; Xuan Hu; Jiaying Nie Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi Date: 2020-01-15
Authors: Xuan Hu; Yangyan Yi; Yuanzheng Zhu; Zhaohui Wang; Shu Wu; Jing Zhang; Jiangwen Wang; Jiaying Nie Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi Date: 2019-12-15