Ma Yongbin, Zhao Leyu, Zhou Dan, Li Tao, Feng Yuhui, Yao Xin, Zhao Kai
Department of Central Laboratory, Jintan Hospital, Jiangsu University, Jintan 213200, China.
Department of Clinical Laboratory and Transfusion Medicine Laboratory, School of Medicine, Jiangsu University, Zhenjiang 212001, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023 Dec;39(12):1069-1073.
Objective To establish an efficient method for isolating migrasomes from RAW264.7 macrophages and identifying these isolated migrasomes. Methods Scanning electron microscopy was used to observe the morphological characteristics of migrasomes produced by RAW264.7 cells. A 0.45 μm filter was employed for reverse filtration and elution to isolate the migrasomes. The morphological characteristics of the migrasomes were then observed using transmission electron microscopy. Western blot analysis was performed to determine the expression of characteristic markers of the migrasomes. The RNA carried by the migrasomes was analysed by using LabChip bioanalyzer. Results Scanning electron microscopy revealed that the migrasomes, with membranous structures, were attached to the tip or bifurcation of the retraction fiber formed in the tail of RAW264.7 cells. Transmission electron microscopy showed that the isolated migrasomes had a typical oval vesicle-like structure with wrinkled membrane surfaces. Western blot analysis confirmed the expression of the characteristic markers phosphatidylinositol glycan anchor biosynthesis class K (PIGK), epidermal growth factor domain-specific O-linked N-acetylglucosamine transferase (EOGT) and tetraspanin 4 (TSPAN4) in the migrasomes, while the EV (extracellular vesicle) markers tumor susceptibility gene 101 (TSG101) and Arabidopsis homolog of apoptosis-linked gene 2-interacting protein X (ALIX) were not detected. Furthermore, the isolated migrasomes were found to be rich in small RNA, which were approximately 25-200 nt in length. Conclusion A method for the extraction of well-structured and high quality migrasomes from macrophages is established.
目的 建立一种从RAW264.7巨噬细胞中分离迁移体并鉴定所分离迁移体的有效方法。方法 利用扫描电子显微镜观察RAW264.7细胞产生的迁移体的形态特征。采用0.45μm滤膜进行反向过滤和洗脱以分离迁移体。然后使用透射电子显微镜观察迁移体的形态特征。进行蛋白质免疫印迹分析以确定迁移体特征性标志物的表达。使用LabChip生物分析仪分析迁移体携带的RNA。结果 扫描电子显微镜显示，迁移体具有膜结构，附着于RAW264.7细胞尾部形成的收缩纤维的末端或分支处。透射电子显微镜显示，分离的迁移体具有典型的椭圆形囊泡样结构，膜表面有褶皱。蛋白质免疫印迹分析证实迁移体中磷脂酰肌醇聚糖锚定生物合成K类（PIGK）、表皮生长因子结构域特异性O-连接N-乙酰葡糖胺转移酶（EOGT）和四跨膜蛋白4（TSPAN4）等特征性标志物的表达，而未检测到细胞外囊泡（EV）标志物肿瘤易感基因101（TSG101）和凋亡相关基因2相互作用蛋白X的拟南芥同源物（ALIX）。此外，发现分离的迁移体富含小RNA，其长度约为25 - 200 nt。结论 建立了一种从巨噬细胞中提取结构良好且高质量迁移体的方法。
