K562细胞分泌的exosome的免疫电镜研究

Observation of K562 cell-secreted exosomes with immunoelectron microscope

目的:通过免疫电镜方法观察K562细胞分泌的 exosome,探讨其潜在的临床应用价值.方法:采用离心超滤和 蔗糖密度梯度离心等方法分离出K562细胞释放的exosome, 经透射电镜观察其超微结构.采用固相免疫电镜法(SPIEM) 制备exosome的HSP70,ICAM 1及ABL免疫电镜标本.结果: K562细胞分泌的exosome为直径50～100nm的膜性微囊结 构,圆形或椭圆形,腔内为低电子密度成分.经抗HSP70, ICAM 1和ABL抗体 胶体金免疫电镜标记,囊外膜及腔内可 见点状、颗粒状电子致密物沉积.结论:离心超滤和蔗糖密度 梯度离心法纯化小量exosome方法可行,K562细胞分泌的ex osome表达HSP70,ICAM 1和ABL蛋白,具备免疫原性.

AIM: To observe K562 cell-secreted exosomes under immunoelectron microscope and to discuss their potential clinical value. METHODS: Exosomes were purified by ultrafiltration centrifugation and sucrose gradient ultracentrifugation. Electron microscopy was used to observe their ultrastructures. The expression of HSP70, ICAM-1 and ABL were investigated by solid phase immunoelectron microscopic method (SPIEM). RESULTS: Transmission electron microscopy revealed that the exosomes from K562 cell culture supernatants were 50 to 100 nm membrane vesicles. They were round or ellipse in shape. Cavum had low electron-density components, which were labeled by colloidal gold HSP70, ICAM-1 and ABL. Immune electron microscopy showed the deposition of electron particles in the extramembrane and cavum of the vesicles. CONCLUSION: Ultrafiltration centrifugation and sucrose gradient ultracentrifugation can be used to purify K562 cells-secreted exosomes. The exosomes express HSP70, ICAM-1 and ABL protein and have immunogenicity.