M1巨噬细胞来源的纳米囊泡通过将M2巨噬细胞转化为M1型巨噬细胞抑制小鼠子宫内膜异位症发展  

Nano-vesicles derived from Ml macrophages affect the development of mouse endometriosis by polarizing M2 macrophages to M1 phenotype

作　　者：蒙秋[1] 谢兴润 黄守国[1] MENG Qiu;XIE Xingrun;HUANG Shouguo(Department of Obstetrics and Gynecology,Haikou Hospital of Central South University Xiangya School of Medicine,Haikou 570208,China;Department of Radiology,Haikou Hospital of Central South University Xiangya School of Medicine,Haikou 570208,China)

机构地区：[1]中南大学湘雅医学院附属海口医院妇产科,海南海口570208 [2]中南大学湘雅医学院附属海口医院放射科,海南海口570208

出　　处：《细胞与分子免疫学杂志》2023年第9期807-815,共9页Chinese Journal of Cellular and Molecular Immunology

基　　金：海南省自然科学基金(821QN423)。

摘　　要：目的探究M1巨噬细胞来源的纳米囊泡(M1-NV)治疗子宫内膜异位症(EMS)的有效性和关键机制。方法通过差速离心法从巨噬细胞培养上清液中分离细胞外囊泡(EV)。免疫荧光细胞化学染色检测波形蛋白(vimentin)、CD31和F4/80表达分别鉴定EMS患者子宫内膜基质细胞(EMS-ESC)、人脐静脉内皮细胞(HUVEC)、极化的腹膜巨噬细胞。通过在0~4℃环境下注射器吸取细胞悬液后,通过(5、1、0.4、0.22)μm聚碳酸酯膜过滤器进行过滤制备M1-NV。流式细胞术分析RAW264.7小鼠腹腔巨噬细胞的体外极化情况、反转录PCR检测其血管内皮生长因子(VEGF)、CD86、白细胞介素6(IL-6)、IL-1β和肿瘤坏死因子α(TNF-α)、精氨酸酶1(Arg1)、CD163、CD206和IL-10的表达。将PKH67标记的M1-NV在与EMS-ESC、HUVEC和巨噬细胞共同培养,通过小管形成实验检测M1-NV对HUVEC小管形成的影响、通过Transwell^(TM)侵袭和迁移实验检测EM-ESC在迁移和浸润能力方面的变化。结果通过对NV内含物的检测发现NV中的蛋白质和其他生物活性颗粒含量远多于来自相同数量的EV;与EMS-ESC、HUVEC和巨噬细胞共同培养时,PKH67标记的M1-NV在均被细胞摄取内化。与M0-NV处理相比,20μg/mL M1-NV就能够有效地将M2巨噬细胞重新编码为M1巨噬细胞。与空白组和M0-NV组相比,M1-NV处理后的EMS-ESC侵袭和迁移明显减少、HUVEC的小管形成减少,而用M2-NV处理的EMS-ESC则相反。结论M1-NV可以将M2巨噬细胞重新编码为M1巨噬细胞,抑制EMS-ESC的侵袭、迁移并阻碍血管形成,进而阻碍EMS的发生和发展。Objective To explore whether nano-vesicles derived from M1 macrophages(M1-NVs)can reprogram M2 macrophages into M1 phenotype and further affect the development of endometriosis(EMS).Methods Extracellular vesicles(EVs)were isolated from macrophage culture supernatant by differential centrifugation.Immunofluorescence cytochemistry was used to detect the expression of vimentin,CD31 and F4/80 to identify endometrial stromal cells(EMS-ESCs),HUVECs and polarized peritoneal macrophages of EMS patients.M1-NVs were prepared by filtering cell suspension through(5,1,0.4,0.22)μm polycarbonate membrane filters after syringe aspiration at 0-4℃.Flow cytometry was used to analyze the polarization of RAW264.7 mouse peritoneal macrophages in vitro,and reverse transcription PCR(RT-qPCR)was employed to detect mRNA expression of VEGF,CD86,interleukin-6(IL-6),IL-1β,tumor necrosis factorα(TNF-α),arginase1(Arg1),CD163,CD206,and IL-10.PKH67-labeled M1-NVs were co-cultured with EMS-ESCs,HUVECs and macrophages.And tubule formation experiments were conducted to assess the impact of M1-NVs on the tubule formation of HUVECs.Transwell^(TM)invasion and migration assays were employed to evaluate changes in the migration and invasion abilities of EMS-ESCs.Results By monitoring the contents of NVs,it was found that NVs contained much more protein and other bioactive particles than the same amount of EVs;immunofluorescence staining results showed PKH67 labeled M1-NVs were internalized by EMS-ESCs,HUVECs and macrophages when co-cultured.The results of flow cytometry and RT-qPCR multi-target analysis showed that after treatment with diferent concentrations of M1-NVs or MO-NVs,20μg/mL of M1-NVs could effectively reprogram M2 macrophages into M1 macrophages compared with MO-NVs.Transewell^(TM)results showed that compared with the blank group and Mo-NVs group,the number of EMS-ESCs migrating from the upper chamber to the lower chamber after M1-NV treatment was significantly reduced,while the number of EMS-ESCs treated with M2NVs increased signifi

关 键 词：子宫内膜异位症 巨噬细胞极化 细胞外囊泡 纳米囊泡 

分 类 号：R711.71[医药卫生—妇产科学] R392.12[医药卫生—临床医学] R271.1