摘要
目的:探讨中性粒细胞外泌体调控巨噬细胞凋亡的可能机制。方法:采用脂多糖诱导中性粒细胞,ELISA检测炎症因子,电镜鉴定外泌体形态,RT‑PCR检测外泌体中miR‑15a‑5p表达;并采用中性粒细胞外泌体与miR‑15a‑5p模拟物分别处理raw267.4巨噬细胞,CCK8检测细胞活力,流式检测细胞凋亡;预测并采用双荧光素酶实验验证miR‑15a‑5p与BCL2L2结合位点;RT‑PCR与Western blot验证miR‑15a‑5p对BCL2L2表达调控。结果:脂多糖诱导的中性粒细胞炎症因子IL‑2、IL‑6、IL‑10与TNF‑α显著升高,电镜观察到外泌体形态特征,miR‑15a‑5p在脂多糖诱导的中性粒细胞外泌体中显著高表达;脂多糖诱导的中性粒细胞外泌体与miR‑15a‑5p模拟物均可以促进raw267.4巨噬细胞凋亡,抑制其细胞活力;Targetscan数据库预测miR‑15a‑5p与BCL2L2具有结合位点,双荧光素酶实验验证miR‑15a‑5p与BCL2L2通过结合位点结合;miR‑15a‑5p模拟物转染raw267.4巨噬细胞后抑制BCL2L2 mRNA与蛋白表达。结论:炎症状态中性粒细胞,可能通过分泌包含miR‑15a‑5p的外泌体,通过靶向抑制BCL2L2进而促进raw267.4巨噬细胞凋亡。这可能为进一步理解中性粒细胞与巨噬细胞炎症调控分子机制提供理论依据。
Objective:To explore the possible mechanism of neutrophil exosomes regulating macrophage apoptosis.Meth-ods:Neutrophils were induced by lipopolysaccharide,and inflammatory factors were detected by ELISA.The morphology of exo-somes was identified by electron microscope,and the expression of mir-15a-5p in exosomes was detected by RT-PCR;Raw267.4 macrophages was treated with neutrophil exosomes and mir-15a-5p mimic respectively,and CCK8 was used to detect cell viabili-ty,flow cytometry to detect apoptosis.The binding sites of mir-15a-5p and BCL2L2 were predicted and verified by double lucifer-ase experiment;RT-PCR and Western blot verified that mir-15a-5p regulate the expression of BCL2L2.Results:Lipopolysaccha-ride induced neutrophil inflammatory factors IL-2,IL-6,IL-10 and TNF-α.The morphological characteristics of exosomes were observed by electron microscope.Mir-15a-5p was significantly overexpressed in neutrophil exosomes induced by lipopolysaccha-ride;Lipopolysaccharide-induced neutrophil exosomes and mir-15a-5p simulants can promote raw267.4 macrophage apoptosis and inhibit its cell viability;Targetscan database predicted that mir-15a-5p and BCL2L2 had binding sites.Double-luciferase experi-ment verified that mir-15a-5p and BCL2L2 bound through binding sites;Mir-15a-5p mimic was transfected into raw267.4 macro-phages which inhibit the expression of BCL2L2 mRNA and protein.Conclusion:Inflammatory neutrophils may promote raw267.4 by secreting exosomes containing mir-15a-5p and inhibiting BCL2L2 by targeting macrophage apoptosis.This may provide a theo-retical basis for further understanding the molecular mechanism of inflammatory regulation of neutrophils and macrophages.
作者
柯桥
胡龙辉
阮楚峻
黎敏
KE Qiao;HU Long‑hui;RUAN Chu‑jun;LI Min(Emergency and Trauma College of Hainan Medical University,Haikou 570311,China;Emergency Center of Hainan Medical University Affiliated Hainan Hospital,Haikou 570311,China;Key Laboratory of Emergency and Trauma,Ministry of Education,Haikou 570311,China)
出处
《海南医学院学报》
2023年第19期1441-1447,共7页
Journal of Hainan Medical University
基金
国家自然科学基金项目(82060357)
海南省自然科学基金(2019RC370820RC757)
海南省卫生健康委员会研究项目(20A200021)。