人绒毛膜滋养层细胞来源含miR-155外泌体对人脐静脉内皮细胞增殖及成管能力的影响  

Effect of human chorionic trophoblast cells-derived miR-155-containing exosomes on the proliferation and tube-forming capacity of human umbilical vein endothelial cells

作　　者：谈爽 赵静 马媛 杨洋 Tan Shuang;Zhao Jing;Ma Yuan;Yang Yang(Second Clinical Medical College,Shaanxi University of Chinese Medicine,Xianyang Shaanxi 712046;Center of Reproductive Medicine,Xi'an People's Hospital(Xian Fourth Hospital),Xi'an Shaanxi 710004;Center of Reproductive Medicine,Tangdu Hospital,Air Force Medical University,Xian Shaanxi 710038,P.R.China)

机构地区：[1]陕西中医药大学第二临床医学院,陕西咸阳712046 [2]西安市人民医院(西安市第四医院)生殖医学中心,陕西西安7100044 [3]空军军医大学唐都医院妇产科生殖医学中心,陕西西安710038

出　　处：《中国计划生育和妇产科》2024年第8期33-37,56,I0001,I0002,共8页Chinese Journal of Family Planning & Gynecotokology

基　　金：陕西省重点研发计划项目(项目编号:2023-YBSF-496);西安市科技计划项目(项目编号:23YXYJ0077);陕西省卫生健康科研基金项目(项目编号:2022D062);西安市卫生健康委员会科研项目(项目编号:2022ms04);西安市人民医院(西安市第四医院)科研孵化基金资助课题(项目编号:ZD-9)。

摘　　要：目的构建含miR-155的人绒毛膜滋养层细胞来源外泌体,探讨miR-155通过外泌体携带对人脐静脉内皮细胞(HUVECs)增殖及成管能力的影响。方法2023年1月至2023年4月于西安市人民医院(西安第四医院)收集人体正常早孕流产绒毛组织进行人绒毛膜滋养层细胞原代培养,并提取、收集细胞上清中的外泌体;通过透射电镜、纳米粒子追踪分析(NTA)、Western-blot鉴定外泌体;通过药载技术构建含miR-155模拟物(Exo-miR-155 mimics)、抑制物(Exo-miR-155 inhibitor)以及对照物(Exo-NC)的外泌体;q-PCR检测外泌体中miR-155表达;将构建好的外泌体分别与HUVECs共孵育,荧光显微镜检测外泌体摄入情况;CCK-8、成管实验检测摄取外泌体后各组HUVECs细胞增殖、成管能力;q-PCR、Western-blot检测HUVECs中CYR61、VEGF表达水平。结果分离培养的人绒毛膜滋养层细胞能够显著表达CK-7;人绒毛膜滋养层细胞培养液中提取的外泌体具有典型的形态、粒径并表达外泌体特征性蛋白CD9、CD63;载入外泌体中的miR-155表达与抑制效果显著;与摄取Exo-NC后的HUVECs相比,摄取Exo-miR-155 mimics后的HUVECs增殖与成管能力均下降;且细胞中CYR61 mRNA、VEGF mRNA及蛋白表达水平均降低。结论miR-155可通过滋养层细胞来源外泌体的携带影响HUVECs增殖及成管能力,以及细胞中血管形成相关蛋白CYR61、VEGF的表达,进而与PE时胎盘功能建立异常和母体全身血管内皮细胞功能障碍相关。Objective To construct miR-155-containing exosomes derived from human chorionic trophoblast cells, and to investigate the effects of miR-155 carried by exosomes on the proliferation and tube-forming ability of human umbilical vein endothelial cells(HUVECs).Methods Fresh aborted tissue with normal development in early pregnancy was collected for primary culture of human chorionic trophoblast cells in Xi'an People's Hospital(Xi′an Fourth Hospital) from January 2023 to April 2023, and exosomes were extracted and collected in the cell supernatant;transmission electron microscopy, nano-particle tracking analysis(NTA), and Western-blot experiments were performed to identify the exosomes. Exosomes containing Exo-miR-155 mimics, Exo-miR-155 inhibitor, and Exo-NC were constructed by drug-loading technology;q-PCR was used to detect the expression of miR-155 in exosomes.The constructed exosomes were then co-incubated with HUVECs respectively, and fluorescence microscopy was used to detect exosome uptake;CCK-8, tube-forming assay were used to detect the proliferation and tube-forming ability of HUVECs cells after exosome uptake. CYR61 and VEGF expression in HUVECs cells were detected by q-PCR and Western-blot.Results Isolated cultured human placental chorionic trophoblast cells were able to significantly express CK-7;exosomes extracted from human placental chorionic trophoblast cell cultures had a typical morphology, particle size and expressed the exosomel characteristic proteins CD9 and CD63;the expression and inhibition effect of miR-155 in the exosomes were significant;compared with HUVECs after uptake of Exo-NC, HUVECs proliferation and tube-forming ability were decreased after uptake of Exo-miR-155 mimics;and the expression of CYR61 mRNA, VEGF mRNA and protein expression were reduced in cells. Conclusion The carriage of miR-155 by trophoblast-derived exosomes can affect the ability of HUVECs to proliferate and form tubes, as well as the expression of angiogenesis-associated proteins CYR61 and VEGF in the cells, which

关 键 词：微小RNA 外泌体 脐静脉内皮细胞 滋养层细胞 

分 类 号：R714.24[医药卫生—妇产科学]