Effect of exosomes from human adipose-derived stem cells on wound healing and migration potential of HaCaT cells: a preliminary experimental study
10.3760/cma.j.issn.0412-4030.2019.09.006
- VernacularTitle: 人脂肪干细胞源外泌体对HaCaT细胞损伤修复及迁移功能影响的初步实验研究
- Author:
Yuanyuan ZHANG
1
;
Yue ZENG
2
;
Yanxia ZHU
2
;
Cuihong LIAN
1
Author Information
1. Department of Dermatology, The First Affiliated Hospital of Shenzhen University, The Second People′s Hospital of Shenzhen, Shenzhen 518035, China
2. Department of Medical Cell Biology and Genetics, Shenzhen University Health Science Center, Shenzhen 518000, China
- Publication Type:Journal Article
- Keywords:
Stem cells;
Subcutaneous fat;
Exosomes;
Keratinocytes;
Wound healing;
Cell migration assays
- From:
Chinese Journal of Dermatology
2019;52(9):616-623
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To preliminarily evaluate the effect of exosomes from human adipose-derived stem cells (hADSCs) on the migration of epidermal cells.
Methods:hADSCs were isolated from adipose tissues obtained from a healthy woman after liposuction, and subjected to culture. Exosomes were collected from the culture supernatant of hADSCs by using a modified method combining ultracentrifugation with ultrafiltration, and identified and isolated by transmission electron microscopy (TEM) , dynamic light scattering (DLS) and Western blot analysis. Some cultured HaCaT cells were divided into several groups to be treated with hydrogen peroxide (H2O2) at different concentrations of 0, 50, 100, 200, 250, 300, 400, 500, 600, 800 μmol/L for 1 hour, and cell counting kit-8 (CCK8) assay was performed to evaluate the effect of H2O2 on the survival rate of HaCaT cells. Some HaCaT cells were classified into 2 groups to be pretreated with phosphate-buffered saline (PBS) (normal group) or 100 μmol/L H2O2 (injury group) for 0.5 hour; then, HaCaT cells in the 2 groups were separately divided into treatment group and control group to be co-cultured with exosomes or not. Confocal fluorescence microscopy was conducted to confirm the uptake of PKH26-labelled exosomes by HaCaT cells, scratch assay to estimate the wound healing potential, and Transwell assay to evaluate the migratory activity of HaCaT cells. Statistical analysis was carried out by using one-way analysis of variance for comparison among groups, least significant difference (LSD) -t test for multiple comparisons, and Spearman correlation analysis for analyzing correlations.
Results:TEM showed that the exosomes isolated from hADSCs were saucer-like nanovesicles with diameters of 60-80 nm. DLS revealed that the purity of the isolated exosomes was 65.88%, and they were stained positively for CD63, Alix and TSG101, which coincided with the basic characteristics of exosomes. CCK8 assay showed that survival rates of HaCaT cells gradually decreased along with the increase of H2O2 concentrations after 1-hour treatment, and were negatively correlated with the concentration of H2O2 (r= -0.91, P < 0.01) . Confocal fluorescence microscopy showed that the isolated exosomes could be endocytosed into impaired HaCaT cells. Scratch assay showed that the gap-filling rates at 12 hours were 40.26% ± 0.64%, 69.57% ± 0.69%, 32.28% ± 0.31% and 69.62% ± 1.68% in the normal control group, normal treatment group, injury control group and injury treatment group respectively, and the injury treatment group showed a significantly increased gap-filling rate compared with the injury control group (t = 37.33, P < 0.01) . Transwell assay showed that the number of migratory cells per × 10 field was 20.85 ± 4.84, 44.8 ± 5.24, 14.95 ± 2.58 and 40.05 ± 7.66 in the normal control group, normal treatment group, injury control group and injury treatment group respectively, and was significantly larger in the injury treatment group than in the injury control group (t = 25.10, P < 0.01) .
Conclusion:Exosomes isolated from hADSCs can improve the migration of HaCaT cells after oxidative injury.
