目的探讨间充质干细胞（MSC）来源的细胞外囊泡（EV）（MSC-EV）在脓毒症相关急性肺损伤（SI-ALI）中对中性粒细胞胞外诱捕网（NET）的影响及其相关机制。
方法将提取的MSC-EV进行表征鉴定，采用纳米流式检测仪检测其粒径范围，电镜检测其形态，免疫印迹法（Western blot）检测EV标记物[凋亡诱导因子6相互作用蛋白抗体（Alix）、溶酶体相关膜蛋白3（CD63）、细胞分化抗原9（CD9）、钙联蛋白（Calnexin）]。将18只野生型C57BL/6J小鼠按随机数字表法分为3组（每组6只）：假手术组（S组）、盲肠结扎穿孔术（CLP）组、治疗组（CME组）。S组小鼠开腹后关腹，CLP组进行CLP，CME组CLP前1 h尾静脉注射MSC-EV（200 μg/只）；苏木精-伊红（H-E）染色比较3组小鼠肺组织病理改变，免疫荧光染色法比较肺组织髓过氧化物酶（MPO）、瓜氨酸化组蛋白H3（CitH3）水平。取人外周血中的中性粒细胞按随机数字表法分为3组：对照组（C组）、丙二醇甲醚醋酸酯组（PMA组）、细胞治疗组（PME组）。C组给予磷酸盐缓冲液（PBS）对照处理，PMA组给予PMA 50 nmol/L，PME组在PMA刺激前1 h给予MSC-EV 20 mg/L；免疫荧光染色法比较3组细胞CitH3水平，活性氧（ROS）染色法比较细胞ROS水平。
结果纳米流式检测结果显示MSC-EV粒径范围为60~100 nm；透射电镜显示杯状的囊泡图像；Western blot显示MSC-EV表达EV的标志蛋白Alix、CD63、CD9，不表达Calnexin。与S组比较，CLP组肺组织肺泡壁较厚、出血较多、炎症细胞浸润较多，肺组织MPO、CitH3水平较高（均 P<0.05）；与CLP组比较，CME组肺组织肺泡壁较薄、出血较少、炎症细胞浸润较少，肺组织MPO、CitH3水平较低（均 P<0.05）。与C组比较，PMA组CitH3、ROS水平较高（均 P<0.05）；与PMA组比较，PME组CitH3、ROS水平较低（均 P<0.05）。
结论MSC-EV可以通过减少ROS从而抑制NET生成，进而缓解SI-ALI。
ObjectiveTo investigate the effect of mesenchymal stem cell-derived extracellular vesicles (MSC-EV) on neutrophil extracellular trap (NET) in sepsis-induced acute lung injury (SI-ALI) and the underlying mechanisms.
MethodsFirst, the extracted MSC-EV were characterized: their size range was detected using nano-flow cytometry, morphology was observed by electron microscopy, and EV markers [Apoptosis linked gene 2 interacting protein X (Alix), CD63, CD9, and Calnexin] were examined by Western blot. Eighteen wild-type C57BL/6J mice were divided into three groups: a sham-operated (S) group, a cecal ligation and puncture (CLP) group, and a treatment (CME) group, with six mice in each group, according to random number table method. Mice in the S group underwent laparotomy without further treatment, followed by wound closure; those in the CLP group received CLP treatment; and the CME group was injected via the tail vein with MSC-EV (200 μg/mouse) 1 h before CLP. Their lung pathologica changes was compared among the three groups by hematoxylin-eosin (H-E) staining. The levels of myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) in lung tissue were detected by immunofluorescence staining. Neutrophils in human peripheral blood samples were divided into three groups: a control (C) group, a phorbol myristate acetate (PMA) group, and a cell therapy (PME) group, according to random number table method. The C group was treated with phosphate-buffered saline (PBS) as a control; the PMA group was treated with PMA 50 nmol/L; and the PME group was treated with MSC-EV 20 mg/L 1 h before PMA stimulation. The levels of CitH3 in the three cell groups were compared by immunofluorescence staining, and reactive oxygen species (ROS) levels were compared using ROS staining.
ResultsNano-flow cytometry showed the MSC-EV particle size range was 60-100 nm, transmission electron microscopy showed cup-shaped vesicles, and Western blot showed that MSC-EV expressed EV markers Alix, CD63, CD9, but not Calnexin. The CLP group showed thicker alveolar walls, more hemorrhage, and more inflammatory cell infiltration in lung tissue, as well as higher MPO and CitH3 levels than the S group (all P<0.05). Compared with the CLP group, the CME group showed decreases in alveolar walls, hemorrhage, and inflammatory cell infiltration in lung tissue, as well as MPO and CitH3 levels (all P<0.05). Compared with the C group, the PMA group showed increased CitH3 and ROS levels (all P<0.05). Compared with the PMA group, the PME group presented reduced CitH3 and ROS levels (all P<0.05).
ConclusionsMSC-EV can inhibit NET formation by reducing ROS, thereby alleviating SI-ALI.
邵毓雯,吴丹,施雨鑫,等. MSC-EV通过抑制中性粒细胞胞外诱捕网的生成减轻脓毒症相关急性肺损伤[J]. 国际麻醉学与复苏杂志,2024,45(11)：1121-1126.
DOI:10.3760/cma.j.cn321761-20240720-01145版权归中华医学会所有。
未经授权，不得转载、摘编本刊文章，不得使用本刊的版式设计。
除非特别声明，本刊刊出的所有文章不代表中华医学会和本刊编委会的观点。
邵毓雯：研究操作、数据整理、论文撰写；吴丹、施雨鑫、王杨寒召：研究操作、统计分析；张浩：研究指导、论文修改；缪长虹：研究指导、经费支持
文档即答
智问智答
机器翻译
你好，我可以帮助您更好的了解本文，请向我提问您关注的问题。
