The effect and mechanism of exosomes derived from human amniotic epithelial cells on the proliferation and migration of HaCaT in high glucose environment
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摘要:
目的 探讨人羊膜上皮细胞外泌体（hAEC-Exo）对高糖环境下HaCaT增殖和迁移的作用及其相关机制。 方法 采用实验研究方法。取2019年1—6月于福建医科大学附属协和医院妇产科10名足月分娩健康孕妇羊膜组织，分离原代人羊膜上皮细胞（hAEC）。观察培养第2、4、7天原代hAEC生长状态和形态改变，采用流式细胞术检测细胞表面标志物CD73、CD90、CD29、CD34及人白细胞抗原DR（HLA-DR）的表达，取第2~4代细胞用于后续实验。超速离心法分离hAEC-Exo。将HaCaT与hAEC-Exo共培养3 h，采用倒置荧光显微镜观察HaCaT对hAEC-Exo的摄取情况。取HaCaT，分为磷酸盐缓冲液（PBS）组、hAEC-Exo组和二甲基亚砜（DMSO）+PBS组、DMSO+hAEC-Exo组、LY294002+hAEC-Exo组，每组3孔，并进行相应处理，采用细胞计数试剂盒8（CCK-8）法检测培养0（即刻）、12、24、36、48、60 h细胞增殖活力；划痕试验检测划痕后0、24、48、72 h划痕愈合情况，并计算划痕愈合率；Transwell实验检测培养48 h穿膜细胞数；蛋白质印迹法检测培养24 h后磷脂酰肌醇3-激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白（PI3K-Akt-mTOR）通路相关的哺乳动物雷帕霉素靶蛋白（mTOR）、磷酸化mTOR（p-mTOR）、蛋白激酶B（Akt）、磷酸化Akt（p-Akt）的蛋白表达。对数据行重复测量方差分析、单因素方差分析及独立样本t检验。 结果 原代hAEC培养第2天多数呈卵圆形，大小均一；培养第4、7天，细胞形态呈典型的鹅卵石样单层排列。原代hAEC高表达间充质干细胞表面标志物CD73、CD90及CD29，不表达或低表达造血干细胞相关表面标志物CD34及HLA-DR。培养3 h，hAEC-Exo成功被HaCaT内吞入细胞质，并聚集于细胞核周围。培养12、24、36、48、60 h，hAEC-Exo组HaCaT增殖活力明显高于PBS组（t=3.691、10.861、12.121、10.531、14.931，P<0.01）。划痕后24、48、72 h，PBS组HaCaT划痕愈合率明显低于hAEC-Exo组（t=3.342、6.427、5.485，P<0.05或P<0.01）。培养48 h，hAEC-Exo组HaCaT穿膜数显著多于PBS组（t=5.385，P<0.01）。培养24 h，hAEC-Exo组HaCaT中p-mTOR和p-Akt蛋白表达量明显高于PBS组（t=4.240、5.586，P<0.01），2组HaCaT中mTOR和Akt蛋白表达量相近（P>0.05）。培养24 h，DMSO+hAEC-Exo组HaCaT中p-mTOR和p-Akt的蛋白表达量明显高于DMSO+PBS组（t=6.155、8.338，P<0.01）和LY294002+hAEC-Exo组（t=5.030、3.960，P<0.01），3组HaCaT中mTOR和Akt蛋白表达量相近（P>0.05）。DMSO+hAEC-Exo组HaCaT培养12、24、36、48、60 h增殖活力为0.78±0.05、1.23±0.07、1.60±0.09、1.86±0.09、2.03±0.08，明显高于DMSO+PBS组的0.46±0.04、0.69±0.07、0.98±0.08、1.16±0.08、1.26±0.11（t=4.376、7.398、8.488、9.766、10.730，P<0.01）；DMSO+hAEC-Exo组HaCaT培养24、36、48、60 h增殖活力明显高于LY294002+hAEC-Exo组的0.96±0.09、1.20±0.08、1.39±0.08、1.55±0.10（t=3.639、5.447、6.605、6.693，P<0.05或P<0.01）。DMSO+hAEC-Exo组HaCaT划痕后24、48、72 h划痕愈合率明显高于DMSO+PBS组（t=4.003、6.349、7.714，P<0.01）和LY294002+hAEC-Exo组（t=3.805、4.676、4.067，P<0.05或P<0.01）。培养48 h，DMSO+hAEC-Exo组HaCaT穿膜数明显多于DMSO+PBS组和LY294002+hAEC-Exo组（t=7.464、1.232，P<0.01）。 结论 PI3K-Akt-mTOR通路介导hAEC-Exo促进高糖环境下HaCaT增殖和迁移。 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. -
1 原代人羊膜上皮细胞（hAEC）培养第2、4、7天形态观察 倒置荧光显微镜×100。1A.培养第2天，hAEC贴壁生长，形态、大小均一；1B、1C.分别为培养第4、7天，hAEC增殖活跃，细胞密度增加，呈典型的鹅卵石样单层排列
2 人羊膜上皮细胞外泌体（hAEC-Exo）与HaCaT共培养3 h，hAEC-Exo被HaCaT内吞入细胞质，并聚集于细胞核周围 PKH26-4',6-二脒基-2-苯基吲哚×200
注：红色指示外泌体，蓝色指示细胞核
3 2组HaCaT划痕后各时间点划痕面积 倒置荧光显微镜×100。3A、3B、3C、3D.分别为PBS组划痕后0（即刻）、24、48、72 h的划痕面积；3E、3F、3G、3H.分别为hAEC-Exo组划痕后0、24、48、72 h的划痕面积，图3F、3G、3H划痕面积分别明显小于图3B、3C、3D
注：PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体
4 Transwell实验观察2组HaCaT培养48 h穿膜迁移情况 苏木精×50。4A、4B.分别为PBS组和hAEC-Exo组，图4B穿膜细胞数明显多于图4A
注：蓝色指示穿膜细胞；PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体
5 蛋白质印迹法检测2组HaCaT培养24 h PI3K-Akt-mTOR通路相关蛋白的表达水平
注：PI3K为磷脂酰肌醇3-激酶，mTOR为哺乳动物雷帕霉素靶蛋白，p-mTOR为磷酸化mTOR，Akt为蛋白激酶B，p-Akt为磷酸化Akt，GAPDH为3-磷酸甘油醛脱氢酶；1、2分别为磷酸盐缓冲液组和人羊膜上皮细胞外泌体组
6 蛋白质印迹法检测3组HaCaT培养24 h PI3K-Akt-mTOR通路相关蛋白的表达量
注：PI3K为磷脂酰肌醇3-激酶，mTOR为哺乳动物雷帕霉素靶蛋白，p-mTOR为磷酸化mTOR，Akt为蛋白激酶B，p-Akt为磷酸化Akt，GAPDH为3-磷酸甘油醛脱氢酶；1、2、3分别为二甲基亚砜（DMSO）+磷酸盐缓冲液组，DMSO+人羊膜上皮细胞外泌体（hAEC-Exo）组，LY294002+hAEC-Exo组
7 3组HaCaT划痕后各时间点划痕面积。7A、7B、7C、7D.分别为DMSO+PBS组划痕后0（即刻）、24、48、72 h的划痕面积；7E、7F、7G、7H.分别为DMSO+hAEC-Exo组划痕后0、24、48、72 h的划痕面积，图7F、7G、7H划痕面积明显小于图7B、7C、7D；7I、7J、7K、7L.分别为LY294002+hAEC-Exo组划痕后0、24、48、72 h的划痕面积，图7J、7K、7L划痕面积明显大于图7F、7G、7H
注：DMSO为二甲基亚砜，PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体
8 Transwell实验观察3组HaCaT培养48 h穿膜迁移情况 苏木精×50。8A、8B、8C.分别为DMSO+PBS组、DMSO+hAEC-Exo组和LY294002+hAEC-Exo组，图8B穿膜迁移细胞数明显多于图8A、8C
注：图中蓝色指示穿膜细胞；DMSO为二甲基亚砜, PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体
表 1 2组HaCaT培养各时间点增殖活力比较（
组别 样本数 12 h 24 h 36 h 48 h 60 h PBS组 3 0.273±0.025 0.386±0.028 0.494±0.030 0.789±0.060 0.943±0.040 hAEC-Exo组 3 0.404±0.030 0.770±0.041 0.923±0.026 1.161±0.042 1.471±0.038 t值 3.691 10.861 12.121 10.531 14.931 P值 <0.01 <0.01 <0.01 <0.01 <0.01 注：PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体；处理因素主效应，F=783.2，P<0.01；时间因素主效应，F=494.7，P<0.01；两者交互作用，F=27.0，P<0.01 
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表2 2组HaCaT划痕后各时间点划痕愈合率比较
表2. （%，x¯±s）
组别 样本数 24 h 48 h 72 h PBS组 3 22.6±2.4 42.8±4.0 74.5±4.5 hAEC-Exo组 3 34.7±4.9 66.1±4.8 94.4±4.1 t值 3.342 6.427 5.485 P值 <0.05 <0.01 <0.01 注：PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体；处理因素主效应，F=692.7，P<0.01；时间因素主效应，F=25.4，P<0.01；两者交互作用，F=14.4，P<0.01
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表3 2组HaCaT培养24 h PI3K-Akt-mTOR通路相关蛋白的表达量比较（
组别 样本数 mTOR p-mTOR Akt p-Akt PBS组 3 0.517±0.045 0.267±0.066 0.823±0.068 0.333±0.050 hAEC-Exo组 3 0.537±0.049 0.437±0.046 0.787±0.025 0.610±0.029 t值 0.404 4.240 0.740 5.586 P值 >0.05 <0.01 >0.05 <0.01 注：PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体；PI3K为磷脂酰肌醇3-激酶，Akt为蛋白激酶B，p-Akt为磷酸化Akt，mTOR为哺乳动物雷帕霉素靶蛋白，p-mTOR为磷酸化mTOR
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表4 3组HaCaT中培养24 h PI3K-Akt-mTOR通路相关蛋白的表达量比较（
组别 样本数 mTOR p-mTOR Akt p-Akt DMSO+PBS组 3 0.716±0.017 0.395±0.038 0.859±0.033 0.228±0.028 DMSO+hAEC-Exo组 3 0.716±0.026 0.600±0.054 0.847±0.029 0.505±0.034 LY294002+hAEC-Exo组 3 0.699±0.027 0.433±0.029 0.865±0.039 0.374±0.033 F值 0.4 13.8 0.1 38.3 P值 >0.05 <0.01 >0.05 <0.01 t1值 0.024 6.155 0.353 8.338 P1值 >0.05 <0.01 >0.05 <0.01 t2值 0.501 5.030 0.517 3.960 P2值 >0.05 <0.01 >0.05 <0.01 注：DMSO为二甲基亚砜，PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体，PI3K为磷脂酰肌醇3-激酶，Akt为蛋白激酶B，p-Akt为磷酸化Akt，mTOR为哺乳动物雷帕霉素靶蛋白，p-mTOR为磷酸化mTOR；t1值、P1值，t2值、P2值分别DMSO+hAEC-Exo组与DMSO+PBS组、LY294002+hAEC-Exo组比较所得
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表5 3组HaCaT培养各时间点增殖活力比较（
组别 样本数 12 h 24 h 36 h 48 h 60 h DMSO+PBS组 3 0.46±0.04 0.69±0.07 0.98±0.08 1.16±0.08 1.26±0.11 DMSO+hAEC-Exo组 3 0.78±0.05 1.23±0.07 1.60±0.09 1.86±0.09 2.03±0.08 LY294002+hAEC-Exo组 3 0.59±0.03 0.96±0.09 1.20±0.08 1.39±0.08 1.55±0.10 t1值 4.376 7.398 8.488 9.766 10.730 P1值 <0.01 <0.01 <0.01 <0.01 <0.01 t2值 2.691 3.639 5.447 6.605 6.693 P2值 >0.05 <0.05 <0.01 <0.01 <0.01 注：DMSO为二甲基亚砜，PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体；处理因素主效应，F=141.3，P<0.01；时间因素主效应，F=279.1，P<0.01；两者交互作用，F=8.2，P<0.01；t1值、P1值，t2值、P2值分别DMSO+hAEC-Exo组与DMSO+PBS组、LY294002+hAEC-Exo组比较所得
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表6 3组HaCaT培养各时间点划痕愈合率比较（%，
组别 样本数 24 h 48 h 72 h DMSO+PBS组 3 11.2±1.5 35.2±4.4 71.2±3.4 DMSO+hAEC-Exo组 3 23.0±2.0 54.0±3.9 94.0±2.9 LY294002+hAEC-Exo组 3 11.8±1.8 40.1±2.5 82.0±2.9 t1值 4.003 6.349 7.714 P1值 <0.01 <0.01 <0.01 t2值 3.805 4.676 4.067 P2值 <0.05 <0.01 <0.01 注：DMSO为二甲基亚砜，PBS为磷酸盐缓冲液，hAEC-Exo为人羊膜上皮细胞外泌体；处理因素主效应，F=779.4，P<0.01；时间因素主效应，F=57.1，P<0.01；两者交互作用，F=2.2，P=0.11；t1值、P1值，t2值、P2值分别DMSO+hAEC-Exo组与DMSO+PBS组、LY294002+hAEC-Exo组比较所得
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