摘要
目的:探究蜕膜巨噬细胞源性外泌体miR-146a-5p对子痫前期滋养细胞的作用及其分子机制。方法:收集子痫前期(preeclampsia,PE)患者和正常妊娠女性蜕膜组织,使用密度梯度法与流式细胞术分选获得巨噬细胞,提取蜕膜巨噬细胞源性外泌体;为了鉴定外泌体,采用透射电镜观察结构,western blot验证外泌体CD63蛋白表达;采用CCK-8检测外泌体对滋养细胞活力的影响;Transwell实验检测滋养细胞迁移变化;qPCR检测外泌体中miR-146a-5p的表达;western blot检测滋养细胞中HIF1α蛋白表达;双荧光素酶报告基因检测miR-146a-5p与HIF1α是否存在结合位点。结果:巨噬细胞外泌体呈现直径约30~130 nm的中间凹陷“饼状”结构形态,CD63高表达,符合外泌体特征。与正常组相比,PE组蜕膜巨噬细胞源性外泌体显著降低滋养细胞增殖与迁移(P<0.001)。PE组蜕膜巨噬细胞源性外泌体中miR-146a-5p表达量显著下降,蜕膜巨噬细胞源性外泌体处理滋养细胞后滋养细胞中HIF1α蛋白表达显著升高,miR-146a-5p和HIF1α之间存在靶向结合位点。结论:PE组蜕膜巨噬细胞源性外泌体可以降低滋养细胞增殖与迁移,这可能与外泌体miR-146a-5p表达下降,从而促进滋养细胞HIF1α蛋白表达有关。
Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1α protein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1α by double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1α in trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1α Protein expression of trophoblast cells.
作者
陈芳荣
毛东瑞
陈小菊
CHEN Fang-rong;MAO Dong-rui;CHEN Xiao-ju(Department of Obstetrics,Hainan General Hospital,Hainan Hospital Affiliated to Hainan Medical University,Haikou 570311,China)
出处
《海南医学院学报》
CAS
2023年第4期268-273,共6页
Journal of Hainan Medical University
基金
海南省自然科学基金面上项目(821MS128,822MS164)
海南省人民医院院级国家自然科学基金培育530工程面上项目(2021MSXM04)。