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.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023 Sep;39(9):807-815.
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 DegreesCelsius. 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 factor α (TNF-α), arginase 1 (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 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 that 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 different concentrations of M1-NVs or M0-NVs, 20 μg/mL of M1-NVs could effectively reprogram M2 macrophages into M1 macrophages compared with M0-NVs. Transewell results showed that compared with the blank group and M0-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 significantly. The invasion situation was similar to the migration situation, indicating that M1-NVs directly or indirectly inhibited invasion, migration and tubule formation of EMS-ESCs in vitro. Conclusion M1-NVs reprogrammes M2 macrophages into M1 macrophages by internalization of primary cells and macrophages, thereby inhibiting invasion, migration and angiogenesis of EMS-ESCs, and further hindering the occurrence and development of EMS.
目的 探讨M1巨噬细胞来源的纳米囊泡（M1-NVs）能否将M2巨噬细胞重编程为M1表型，并进一步影响子宫内膜异位症（EMS）的发展。方法 通过差速离心从巨噬细胞培养上清中分离细胞外囊泡（EVs）。采用免疫荧光细胞化学检测波形蛋白、CD31和F4/80的表达，以鉴定EMS患者的子宫内膜基质细胞（EMS-ESCs）、人脐静脉内皮细胞（HUVECs）和极化的腹膜巨噬细胞。在0-4摄氏度下注射器抽吸后，通过（5、1、0.4、0.22）μm聚碳酸酯膜过滤器过滤细胞悬液制备M1-NVs。采用流式细胞术分析RAW264.7小鼠腹膜巨噬细胞的体外极化情况，并采用逆转录聚合酶链反应（RT-qPCR）检测血管内皮生长因子（VEGF）、CD86、白细胞介素-6（IL-6）、IL-1β、肿瘤坏死因子α（TNF-α）、精氨酸酶1（Arg1）、CD163、CD206和IL-10的mRNA表达。将PKH67标记的M1-NVs与EMS-ESCs、HUVECs和巨噬细胞共培养。并进行小管形成实验，以评估M1-NVs对HUVECs小管形成的影响。采用Transwell侵袭和迁移实验评估EMS-ESCs迁移和侵袭能力的变化。结果 通过监测NVs的含量，发现NVs比等量的EVs含有更多的蛋白质和其他生物活性颗粒；免疫荧光染色结果显示，共培养时PKH67标记的M1-NVs被EMS-ESCs、HUVECs和巨噬细胞内化。流式细胞术和RT-qPCR多靶点分析结果显示，用不同浓度的M1-NVs或M0-NVs处理后，与M0-NVs相比，20μg/mL的M1-NVs能有效将M2巨噬细胞重编程为M1巨噬细胞。Transwell结果显示，与空白组和M0-NVs组相比，M1-NV处理后从上层小室迁移到下层小室的EMS-ESCs数量显著减少，而用M2NVs处理的EMS-ESCs数量显著增加。侵袭情况与迁移情况相似，表明M1-NVs在体外直接或间接抑制了EMS-ESCs的侵袭、迁移和小管形成。结论 M1-NVs通过被原代细胞和巨噬细胞内化将M2巨噬细胞重编程为M1巨噬细胞，从而抑制EMS-ESCs的侵袭、迁移和血管生成，进而阻碍EMS的发生发展。
