CP-25调控巨噬细胞来源的外泌体分泌对子宫内膜癌Ishikawa细胞系自噬性细胞死亡的影响  

CP-25 regulates effect of macrophage-derived exosomes on autophagic cell death of endometrial carcinoma Ishikawa cells

作　　者：郑小妹 林叶飞 张韶琼 杨洁 陈曼玲[1] ZHENG Xiaomei;LIN Yefei;ZHANG Shaoqiong;YANG Jie;CHEN Manling(Department of Gynecology,First Affiliated Hospital of Hainan Medical College,Haikou 570100,China)

机构地区：[1]海南医学院第一附属医院妇科,海口570100

出　　处：《中国免疫学杂志》2023年第2期323-329,共7页Chinese Journal of Immunology

基　　金：海南省卫生计生行业科研项目(18A200093)。

摘　　要：目的:探讨芍药苷-6'-O-苯磺酸酯(CP-25)通过调控巨噬细胞来源的外泌体对子宫内膜癌Ishikawa细胞自噬性细胞死亡的影响。方法:使用佛波酯(PMA)将人单核细胞THP-1诱导为巨噬细胞,并提取其外泌体,透射电子显微镜观察外泌体形态,Western blot鉴定外泌体标志蛋白表达;使用不同浓度CP-25(0.1、0.5、1.0μmol/L)干预巨噬细胞外泌体,并与Ishikawa细胞共培养24 h,CCK-8法检测各处理组Ishikawa细胞活性,Transwell小室检测各处理组Ishikawa细胞的迁移与侵袭;以自噬抑制剂3-MA干预细胞,实验分为对照组、3-MA组、Exo-CP-25组、3-MA+Exo-CP-25组,进行对应处理后,Hoechst 33342染色观察各处理组Ishikawa细胞凋亡形态,流式细胞术检测各处理组Ishikawa细胞凋亡率,透射电子显微镜观察各处理组Ishikawa细胞自噬体,免疫荧光染色检测各处理组Ishikawa细胞自噬相关标志物LC3表达,Western blot检测各处理组Ishikawa细胞自噬相关蛋白表达。结果:经鉴定成功分离了诱导后巨噬细胞的外泌体;巨噬细胞来源外泌体与Ishikawa细胞共培养后,细胞活性升高(P<0.05),细胞迁移数与侵袭数均增加(P<0.01);CP-25干预巨噬细胞来源的外泌体与Ishikawa细胞共培养后,细胞活性下降(P<0.05),细胞迁移数与侵袭数均减少(P<0.01);经CP-25干预巨噬细胞来源的外泌体与Ishikawa细胞共培养后,细胞内出现浓缩、碎裂凋亡体,细胞凋亡率增加(P<0.01),LC3绿色荧光表达强度增加(P<0.01),细胞内可见多个由单层膜或双层膜组成的自噬体,同时,LC3-Ⅱ、Beclin-1蛋白表达增加,P62蛋白表达减少,LC3-Ⅱ/LC3-Ⅰ显著上调(P<0.01);而3-MA处理Ishikawa细胞后再用CP-25干预巨噬细胞来源的外泌体与Ishikawa细胞共培养后,细胞核浓缩与碎裂的现象减少,细胞凋亡率下降(P<0.01),LC3绿色荧光表达强度减弱(P<0.01),细胞内自噬体减少,LC3-Ⅱ、Beclin-1蛋白表达减少,P62蛋白表达增加,LC3-Ⅱ/LC3-Ⅰ显著下调(P<0.01)�Objective:To investigate the effect of paeoniflorin-6'-O-benzene sulfonate(CP-25)on autophagic cell death of endometrial cancer Ishikawa cells by regulating exosomes derived from macrophages.Methods:Phorbol ester(PMA)was used to induce human monocyte THP-1 into macrophages,the exosomes were extracted,observed morphology of exosomes with transmission electron microscope,and identified expressions of marker proteins of exosomes by Western blot;different concentrations of CP-25(0.1,0.5,1.0μmol/L)were used to interfere with macrophage exosomes and co-culture with Ishikawa cells for 24 h.CCK-8 method was used to detect activity of Ishikawa cells in each treatment group,Transwell chamber was used to detect migration and invasion of Ishikawa cells in each treatment group;autophagy inhibitor 3-MA was used to intervene cells,experiment was divided into control group,3-MA group,Exo-CP-25 group and 3-MA+Exo-CP-25 group.After corresponding treatment,Hoechst 33342 staining was used to observe apoptotic morphology of Ishikawa cells in each treatment group,flow cytometry was used to detect apoptosis rate of Ishikawa cells in each treatment group,transmission electron microscope was used to observe autophagosomes in Ishikawa cells of each treatment group,immunofluorescence staining was used to detect expression of autophagy-related marker LC3 in Ishikawa cells of each treatment group,and Western blot was used to detect expressions of autophagy-related proteins in Ishikawa cells of each treatment group.Results:After identification,exosomes of the induced macrophages were successfully isolated;after macrophage-derived exosomes were co-cultured with Ishikawa cells,cell viability was increased(P<0.05),and the number of cell migration and invasion increased(P<0.01);after CP-25 interfered with macrophage-derived exosomes and co-cultured with Ishikawa cells,cell viability was decreased(P<0.05),and the number of cell migration and invasion decreased(P<0.01);after CP-25 intervention in macrophage-derived exosomes co-cultured with Ishika

关 键 词：巨噬细胞 外泌体 子宫内膜癌 CP-25 细胞自噬性死亡 

分 类 号：R737.33[医药卫生—肿瘤]