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目的分离K562细胞释放的exosomes,致敏脐血树突细胞(dendritic cell,DCs),观察其对细胞毒性T淋巴细胞(cytotoxic Tlymphocytes,CTLs)的激活效应。方法离心超滤和蔗糖密度梯度离心法分离K562细胞释放的exosomes,固相免疫电镜法(SPIEM)制备exosomes的HSP70、ICAM-1及ABL免疫电镜标本。常规方法从脐血单个核细胞诱导DCs并分离T细胞,将K562细胞来源的exosomes冲击或未冲击的DCs与T细胞共培养。MTT比色法检测体外细胞毒活性。结果K562细胞分泌的exosomes为直径50~100nm的膜性微囊。Exosomes致敏的脐血DCs激活CTLs的能力显著高于肿瘤冻融抗原致敏的DCs组,在效靶比为501时,两组CTLs对K562细胞的杀伤率为(68.4%vs35.3%,P<0.05)。结论K562细胞分泌的exosomes负载脐血DCs后活化CTLs,有抗肿瘤活性。
Objective To isolate exosomes released from K562 cells and sensitize dendritic cells (DCs) to observe the activation of cytotoxic T lymphocytes (CTLs). Methods The exosomes released from K562 cells were isolated by centrifugal ultrafiltration and sucrose density gradient centrifugation. HSP70, ICAM-1 and ABL immunoelectron microscopy were prepared by solid phase immunoelectron microscopy (SPIEM). Conventional methods induce DCs from cord blood mononuclear cells and isolate T cells, co-culturing T cells with or without K562 cell-derived exosomes. MTT colorimetric assay of cytotoxic activity in vitro. Results The exosomes secreted by K562 cells were membranous microcapsules with diameter of 50 ~ 100 nm. Exosomes-sensitized cord blood DCs activated CTLs significantly higher than the ability of tumor freeze-thaw antigen-primed DCs group, the effective target ratio of 501, two groups of CTLs K562 cell killing rate (68.4% vs35.3% P <0.05). Conclusion The exosomes secreted by K562 cells activate CTLs after loading cord blood DCs and have antitumor activity.