外泌体调节复发性流产小鼠子宫内膜细胞上皮间充质转化的作用机制研究  

Exosomes Regulate Epithelial Mesenchymal Transition of Endometrial Cells in Mice with Recurrent Spontaneous Abortion

作　　者：李梦元 李冠杉 韩倩 李思瑶 郑舒畅 辛明蔚[1] 尹晓丹[1] 王景尚[1] 武颖[1] 何军琴[1] LI Mengyuan;LI Guanshan;HAN Qian;LI Siyao;ZHENG Shuchang;XIN Mingwei;YIN Xiaodan;WANG Jingshang;WU Ying;HE Junqin(Department of Traditional Chinese Medicine,Beijing Obstetrics and Gynecology Hospital,Capital Medical University,Beijing Maternal and Child Health Care Hospital,Beijing 100026,China)

机构地区：[1]首都医科大学附属北京妇产医院/北京妇幼保健院中医科,北京市100026

出　　处：《中国全科医学》2025年第8期962-972,共11页Chinese General Practice

基　　金：国家自然科学基金面上资助项目(82074477);北京市自然科学基金面上资助项目(7222268)。

摘　　要：背景复发性流产(RSA)是常见的生殖障碍性疾病,在胚胎植入过程中,子宫内膜发生上皮间充质转化(EMT),RSA的发生与该过程减弱密切相关。子宫内膜来源外泌体及其携带的miRNA参与细胞间通讯,影响细胞EMT,与RSA关系密切,但具体作用机制尚不明确。目的本研究聚焦于外泌体在细胞EMT中的功能,通过实验验证子宫内膜来源外泌体及其分泌的miRNA-221对RSA小鼠子宫内膜细胞EMT的调控作用。方法2021年10月—2022年5月,选取SPF级、健康8周龄雌性小鼠24只,随机选取12只作为正常对照组,剩余12只小鼠制作RSA模型,造模结束后雌雄合笼,妊娠第4日处死雌性小鼠,分离和培养子宫内膜上皮细胞并收集外泌体,通过透射电镜和免疫印迹法(WB)进行外泌体鉴定;通过CCK-8、Transwell和流式细胞术检测外泌体干预前后两组细胞增殖、侵袭、迁移能力;细胞EMT相关蛋白[E-cadherin、N-cadherin、Vimentin、基质金属蛋白酶7(MMP-7)、MMP-9]及外泌体标志蛋白(CD63、CD81、TSG101)的表达;外泌体处理后的细胞EMT标志蛋白(CD24-APC、CD44-FITC)的表达;转染miRNA-221模拟物和抑制物分析外泌体源性miRNA-221对小鼠子宫内膜上皮细胞EMT的作用,并通过WB和双荧光素酶报告基因检测进行分析。结果RSA组小鼠的子宫内膜上皮细胞具有异常的EMT,小鼠子宫内膜上皮细胞能够分泌外泌体。添加外泌体的RSA组细胞增殖率、迁移能力、侵袭能力、CD44^(+)/CD24^(+)表达水平高于细胞上清液RSA组(P<0.05)。与细胞上清液RSA组相比,添加外泌体的RSA组细胞的EMT标志蛋白E-cadherin水平降低,间质样标志蛋白N-cadherin和Vimentin以及基质标志蛋白MMP-7、MMP-9蛋白水平升高(P<0.05)。RSA组miRNA-221表达水平低于正常对照组(P<0.05)。与对照随机miRNA相比,miRNA-221模拟物激活了PI3K-AKT通路、NF-κB通路和p38通路(P<0.05)。与对照随机miRNA相比,miRNA-221模拟物降低了PTEN mRNA的表达水平(PBackground Recurrent spontaneous abortion(RSA)is a common reproductive disorder closely linked with the weakened epithelial-mesenchymal transition(EMT)in the endometrium during embryo implantation.Endometrium-derived exosomes and miRNAs they transport are linked with RSA via the involvement in cellular communication and EMT,although the specific mechanisms have not yet fully revealed.Objective Focusing on the function of exosomes in EMT,this study aims to validate the regulatory role of endometrium-derived exosomes and miRNA-221 they transport in EMT of endometrial cells in RSA mice.Methods From October 2021 to May 2022,a total of 24 eight-week-old female mice in the specific pathogen-free(SPF)level were randomly assigned into the control group(n=12)and RSA group(n=12)for modeling.After RSA modeling,male and female mice were housed together.On the 4th day of pregnancy,female mice were sacrificed for isolating endometrial epithelial cells.They were cultured for extracting exosomes,which were identified by transmission electron microscopy and Western blotting.Cell proliferation,invasion,and migration abilities were evaluated using CCK-8 assay,Transwell assay,and fluorescence-activated cell sorting(FACS)before and after exosome interventions.Expression levels of EMT-related genes[E-cadherin,N-cadherin,Vimentin,matrix metalloproteinase 7(MMP-7),matrix metalloproteinase 9(MMP-9)]and exosome marker proteins[CD63,CD81,tumor susceptibility gene 101(TSG101)]were measured.Expression levels of EMT marker proteins(CD24-APC,CD44-FITC)in exosome-treated cells were analyzed.Transfection of miRNA-221 mimics and inhibitors was performed to analyze the effects of exosome-derived miRNA-221 on EMT of endometrial epithelial cells in RSA mice.Relevant analyses were conducted using Western blot and dual-luciferase reporter assay.Results Abnormal EMT was observed in the endometrial epithelial cells of mice in RSA group,and the mouse endometrial epithelial cells were able to secrete exosomes.The proliferation rate,migration ability,invas

关 键 词：复发性流产 上皮间充质转化 外泌体 miRNA-221 PTEN基因 PI3K-AKT通路 

分 类 号：R714.21[医药卫生—妇产科学]