一种巨噬细胞迁移体分离方法的建立

Establishment of a method for separatingmacroophage migrasomes

目的 建立一种巨噬细胞(RAW264.7)迁移体(migrasomes)的分离方法,并进行迁移体的鉴定。方法 采用扫描电镜观察RAW264.7细胞产生迁移体的形态特征,利用0.45μm滤器反向过滤洗脱的方式分离迁移体,通过透射电镜观察迁移体的形态特征;Western blot法检测迁移体的特征性标志物的表达情况;LabChip生物分析仪分析迁移体携带的RNA情况。结果 扫描电镜观察RAW264.7细胞尾部形成的收缩丝的尖端或分叉处附着具有膜性结构的迁移体;透射电镜观察分离的迁移体呈典型的椭圆形囊泡样结构且膜表面有褶皱;迁移体表达其特征性标志物糖基磷脂酰肌醇锚定生物合成K类(PIGK)、表皮生长因子结构域特异性O-连接的N-乙酰氨基葡萄糖转移酶(EOGT)、四跨膜蛋白家族4(TSPAN4),但不表达细胞外囊泡(EV)的标志物肿瘤易感基因101(TSG101)和凋亡关联基因2相互作用蛋白X拟南芥同源物(ALIX);此外,分离的迁移体携带有丰富的小RNA,长度约为25~200个核苷酸。结论 成功建立了一种从巨噬细胞中提取结构完整且质量较高的迁移体的方法。

Objective To establish an efficient method for isolating migrasomes from RAW264.7 macrophages and identifying these isolated migrasomes.Methods Is Scanning electron microscopy was used to observe the morphological characteristics of migrasomes produced by RAW264.7 cells.A 0.45μm 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.