Roles and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment

Objective: To investigate the roles 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 full-term pregnant women in the Department of Obstetrics and Gynecology of Fujian Medical University Union Hospital from January to June 2019. The primary human amniotic epithelial cells (hAECs) were isolated, and the growth status and morphological change of the primary hAECs on the 2nd, 4th, and 7th day of culture were observed. The 2nd to 4th passages of hAECs were used in the following experiments after being identified by flow cytometry. The hAEC-Exos were separated by ultracentrifugation method. The HaCaT and hAEC-Exos were co-cultured for 3 h, and the uptaken of hAEC-Exos by HaCaT was observed by inverted fluorescence microscope. 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) was used to detect cell proliferation activity at 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), kinase-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 were highly expressed CD73, CD90, and CD29 of mesenchymal stem cell related surface markers, and were with no or low expressions of CD34 and human leukocyte antigen 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 0.05). 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 0.05). 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 higher 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.