Wei P, Xu Z R, Chen Y M, Chen X D, Chen Z H
Department of Burns and Wound Repair, Union Hospital, Fujian Medical University, Fujian Provincial Burn Research Institute, Fujian Burn Medical Center, Fujian Provincial Key Laboratory of Burn and Trauma, Fuzhou 350001, China.
Zhonghua Shao Shang Za Zhi. 2021 Dec 20;37(12):1175-1184. doi: 10.3760/cma.j.cn501120-20210424-00154.
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. 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 test. 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 (=3.691, 10.861, 12.121, 10.531, 14.931, <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 =3.342, 6.427, 5.485, <0.05 or <0.01). After 48 h of culture, the number of transmembrane HaCaT in hAEC-Exos group was significantly more than that in PBS group (=5.385, <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 (=4.240, 5.586, <0.01), while the protein expressions of mTOR and Akt in HaCaT of the two groups were similar (>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 (=6.155, 8.338, <0.01) and LY294002+hAEC-Exos group (=5.030, 3.960, <0.01), while the protein expressions of mTOR and Akt in HaCaT of the three groups were similar (>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 (=4.376, 7.398, 8.488, 9.766, 10.730, <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 (=3.639, 5.447, 6.605, 6.693, <0.05 or <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 (=4.003, 6.349, 7.714, <0.01) and LY294002+hAEC-Exos group (=3.805, 4.676, 4.067, <0.05 or <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 (=7.464, 1.232, <0.01). PI3K-Akt-mTOR pathway can promote the proliferation and migration of HaCaT in high glucose environment by mediating hAEC-Exos.
探讨人羊膜上皮细胞来源的外泌体（hAEC-Exos）在高糖环境下对HaCaT细胞增殖和迁移的影响及机制。采用实验研究方法。收集2019年1月至6月福建医科大学附属协和医院妇产科10例足月分娩的健康孕妇的羊膜组织，分离原代人羊膜上皮细胞（hAECs）。观察原代hAECs培养第2、4、7天的生长状态和形态变化，检测细胞表面标志物CD73、CD90、CD29、CD34和人类白细胞抗原DR（HLA-DR）的表达。后续实验采用第2至4代hAECs。采用超速离心法分离hAEC-Exos。将HaCaT细胞与hAEC-Exos共培养3小时，通过倒置荧光显微镜观察HaCaT细胞对hAEC-Exos的摄取情况。将HaCaT细胞分为磷酸盐缓冲液（PBS）组和hAEC-Exos组，或二甲基亚砜（DMSO）+PBS组、DMSO+hAEC-Exos组和LY294002+hAEC-Exos组，每组设3个复孔，分别进行相应处理。采用细胞计数试剂盒8（CCK-8）法检测培养0（即刻）、12、24、36、48和60小时后细胞的增殖活性。进行划痕实验，检测划痕后0、24、48和72小时的划痕愈合情况，并分别计算划痕愈合率。进行Transwell实验，检测培养48小时后的跨膜细胞数量。采用蛋白质免疫印迹法检测培养24小时后与磷脂酰肌醇3激酶-蛋白激酶B-雷帕霉素靶蛋白（PI3K-Akt-mTOR）通路相关的雷帕霉素靶蛋白（mTOR）、磷酸化mTOR（p-mTOR）、蛋白激酶B（Akt）和磷酸化Akt（p-Akt）的蛋白表达。数据采用重复测量方差分析、单因素方差分析和独立样本检验进行统计学分析。原代hAECs在培养第2天时大多呈椭圆形，大小均匀。在培养第4天和第7天时，hAECs呈典型的鹅卵石样单层排列。原代hAECs高表达间充质干细胞相关表面标志物CD73、CD90和CD29，低表达或不表达造血干细胞相关表面标志物CD34和HLA-DR。共培养3小时后，hAEC-Exos成功被HaCaT细胞内吞进入细胞质并聚集在细胞核周围。培养12、24、36、48和60小时后，hAEC-Exos组HaCaT细胞的增殖活性显著高于PBS组（F = 3.691、10.861、12.121、10.531、14.931，P < 0.01）。划痕后24、48和72小时，PBS组HaCaT细胞的划痕愈合率显著低于hAEC-Exos组（F = 3.342、6.427、5.485，P < 0.05或P < 0.01）。培养48小时后，hAEC-Exos组跨膜HaCaT细胞数量显著多于PBS组（F = 5.385，P < 0.01）。培养24小时后，hAEC-Exos组HaCaT细胞中p-mTOR和p-Akt的蛋白表达显著高于PBS组（F = 4.240、5.586，P < 0.01），而两组HaCaT细胞中mTOR和Akt的蛋白表达相似（P > 0.05）。培养24小时后，DMSO+hAEC-Exos组HaCaT细胞中p-mTOR和p-Akt的蛋白表达显著高于DMSO+PBS组（F = 6.155、8.338，P < 0.01）和LY294002+hAEC-Exos组（F = 5.030、3.960，P < 0.01），而三组HaCaT细胞中mTOR和Akt的蛋白表达相似（P > 0.05）。培养12、24、36、48和60小时后，DMSO+hAEC-Exos组HaCaT细胞的增殖活性分别为0.78±0.05、1.23±0.07、1.60±0.09、1.86±0.09和2.03±0.08，显著高于DMSO+PBS组的0.46±0.04、0.69±0.07、0.98±0.08、1.16±0.08和1.26±0.11（F = 4.376、7.398、8.488、9.766、10.730，P < 0.01）。培养24、36、48和60小时后，DMSO+hAEC-Exos组HaCaT细胞的增殖活性显著高于LY294002+hAEC-Exos组的0.96±0.09、1.20±0.08、1.39±0.08和1.55±0.10（F = 3.639、5.447、6.605、6.693，P < 0.05或P < 0.01）。划痕后24、48和72小时，DMSO+hAEC-Exos组HaCaT细胞的划痕愈合率显著高于DMSO+PBS组（F = 4.003、6.349、7.714，P < 0.01）和LY294002+hAEC-Exos组（F = 3.805、4.676、4.067，P < 0.05或P < 0.01）。培养48小时后，DMSO+hAEC-Exos组跨膜HaCaT细胞数量分别显著多于DMSO+PBS组和LY294002+hAEC-Exos组（F = 7.464、1.232，P < 0.01）。PI3K-Akt-mTOR通路可通过介导hAEC-Exos促进高糖环境下HaCaT细胞的增殖和迁移。
