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  

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

作　　者：卫裴 许钊荣[1] 陈艺敏 陈晓东[1] 陈昭宏[1] Wei Pei;Xu Zhaorong;Chen Yimin;Chen Xiaodong;Chen Zhaohong(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)

机构地区：[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

出　　处：《中华烧伤杂志》2021年第12期1175-1184,共10页Chinese Journal of Burns

基　　金：福建省医疗“创双高”建设项目(闽卫医政〔2021〕76号)。

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

关 键 词：细胞增殖 细胞迁移分析 HACAT 外泌体 人羊膜上皮细胞 磷脂酰肌醇3-激酶-蛋白激酶B-哺乳动物雷帕霉素靶蛋白通路 

分 类 号：R644[医药卫生—外科学] R473.6[医药卫生—临床医学]