The effect and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment
10.3760/cma.j.cn501120-20210424-00154
- VernacularTitle:人羊膜上皮细胞外泌体对高糖环境下HaCaT增殖和迁移的影响及其机制
- Author:
Pei WEI
1
;
Zhaorong XU
;
Yimin CHEN
;
Xiaodong CHEN
;
Zhaohong CHEN
Author Information
1. 福建医科大学附属协和医院烧伤与创面修复科，福建省烧伤研究所，福建省烧伤医学中心，福建省烧创伤重点实验室，福州 350001
- Keywords:
Cell proliferation;
Cell migration assays;
Exosomes;
HaCaT;
Human amniotic epithelial cells;
Phosphatidylinositol 3-kinase-protein kinase B-mammalian targe
- From:
Chinese Journal of Burns
2021;37(12):1175-1184
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect and mechanism of exosomes derived from human amniotic epithelial cells (hAEC-Exos) on the proliferation and migration of HaCaT in high glucose environment.Methods:The experimental research method was adopted. The amniotic membrane tissue was collected from 10 healthy pregnant women at full term delivery in the Department of Obstetrics and Gynecology of Fujian Medical University Union Hospital from January to June 2019, and the primary human amniotic epithelial cells (hAECs) were isolated. The growth status and morphological changes of the primary hAECs on the 2nd, 4th, and 7th day of culture were observed, and the expressions of the cells surface markers of CD73, CD90, CD29, CD34, and human leukocyte antigen DR (HLA-DR). The 2nd to 4th passages of hAECs were used in the following experiments. The hAEC-Exos were separated by ultracentrifugation method. The HaCaT and hAEC-Exos were co-cultured for 3 h, and the uptake of hAEC-Exos by HaCaT was observed by inverted fluorescence microscopy. The HaCaT were divided into phosphate buffer solution (PBS) group and hAEC-Exos group or dimethyl sulfoxide (DMSO)+PBS group, DMSO+hAEC-Exos group, and LY294002+hAEC-Exos group, which were dealt correspondingly, with 3 wells in each group. Cell counting kit 8 (CCK-8) method was used to detect cell proliferation activity after 0 (immediately), 12, 24, 36, 48, and 60 h of culture. The scratch test was conducted to detect the scratch healing at 0, 24, 48, and 72 h after the scratch, and the scratch healing rate was calculated, respectively. The Transwell experiment was conducted to detect the number of transmembrane cells after 48 h of culture. The Western blotting was used to detect the protein expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), protein kinase B (Akt), and phosphorylated Akt (p-Akt) related to phosphatidylinositol 3-kinase-Akt-mTOR (PI3K-Akt-mTOR) pathway after 24 h of culture. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results:Most of the primary hAECs were oval and uniform in size on the 2nd day of culture. The hAECs were arranged in a typical cobblestone-like monolayer on the 4th and 7th day of culture. The primary hAECs highly expressed CD73, CD90, and CD29 of mesenchymal stem cell related surface markers, and were with no or low expressions of CD34 and HLA-DR of hematopoietic stem cell related surface markers. After 3 h of culture, hAEC-Exos were successfully endocytosed by HaCaT into the cytoplasm and gathered around the nucleus. After 12, 24, 36, 48, and 60 h of culture, the proliferation activity of HaCaT in hAEC-Exos group was significantly higher than that in PBS group ( t=3.691, 10.861, 12.121, 10.531, 14.931, P<0.01). At 24, 48, and 72 h after scratch, the scratch healing rates of HaCaT in PBS group were significantly lower than those in hAEC-Exos group (t=3.342, 6.427, 5.485, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in hAEC-Exos group was significantly more than that in PBS group ( t=5.385, P<0.01). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of hAEC-Exos group were significantly higher than those in PBS group ( t=4.240, 5.586, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the two groups were similar ( P>0.05). After 24 h of culture, the protein expressions of p-mTOR and p-Akt in HaCaT of DMSO+hAEC-Exos group were significantly higher than those in DMSO+PBS group ( t=6.155, 8.338, P<0.01) and LY294002+hAEC-Exos group ( t=5.030, 3.960, P<0.01), while the protein expressions of mTOR and Akt in HaCaT of the three groups were similar ( P>0.05). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 12, 24, 36, 48, and 60 h of culture was 0.78±0.05, 1.23±0.07, 1.60±0.09, 1.86±0.09, and 2.03±0.08, which was significantly higher than 0.46±0.04, 0.69±0.07, 0.98±0.08, 1.16±0.08, and 1.26±0.11 in DMSO+PBS group ( t=4.376, 7.398, 8.488, 9.766, 10.730, P<0.01). The proliferation activity of HaCaT in DMSO+hAEC-Exos group at 24, 36, 48, and 60 h was significantly higher than 0.96±0.09, 1.20±0.08, 1.39±0.08, and 1.55±0.10 in LY294002+hAEC-Exos group ( t=3.639, 5.447, 6.605, 6.693, P<0.05 or P<0.01). The scratch healing rates of HaCaT in DMSO+hAEC-Exos group at 24, 48, and 72 h after scratch were significantly higher than those in DMSO+PBS group ( t=4.003, 6.349, 7.714, P<0.01) and LY294002+hAEC-Exos group ( t=3.805, 4.676, 4.067, P<0.05 or P<0.01). After 48 h of culture, the number of transmembrane HaCaT in DMSO+hAEC-Exos group was significantly more than that in DMSO+PBS group and LY294002+hAEC-Exos group, respectively ( t=7.464, 1.232, P<0.01). Conclusions:PI3K-Akt-mTOR pathway can promote the proliferation and migration of HaCaT in high glucose environment by mediating hAEC-Exos.
