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目的:探索白血病细胞来源的胞外体(leukemia cell-derived exosome,LEX)的生物学特性及其致敏DC的抗白血病免疫效应。方法:超速离心分离纯化BCR-ABL阳性的白血病K562细胞来源的胞外体(LEXK562),采用免疫电镜及Westernblotting检测LEXK562中热激蛋白70(heat shock protein 70,HSP70)及BCR-ABL的表达,采用激光扫描共聚焦显微镜及流式细胞术检测LEXK562靶向结合DC的动力学,采用LDH释放法以及小鼠模型体内实验检测LEX及其致敏DC的抗白血病免疫效应。结果:与其他细胞来源的胞外体相似,K562细胞来源的胞外体LEXK562为直径50～100 nm的囊状结构,且表达K562细胞特异性的BCR-ABL和HSP70分子。LEXK562在体外可靶向结合DC,3～4 h到达高峰,并能在DC中稳定存在72 h以上。LEXK562致敏DC(DC/LEXK562)诱导的细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)可有效杀伤K562靶细胞,效靶比为50∶1时,其杀伤活性显著高于LEXK562诱导的CTL[(68.6±5.7)%vs(22.5±2.9)%,P<0.01];体内实验进一步证实,白血病L1210细胞来源的胞外体(LEXL1210)免疫后小鼠接种L1210细胞的成瘤率明显高于LEXL1210致敏DC(DC/LEXL1210)免疫小鼠的成瘤率[(54.17±8.33)%vs(16.67±4.18)%,P<0.05]。结论:LEX表达白血病细胞相关抗原,LEX体外可靶向结合DC,其致敏的DC能诱导更强的抗白血病免疫效应。
Objective: To explore the biological characteristics of leukemia cell-derived exosome (LEX) and its anti-leukemia immunity effect on dendritic cells. Methods: The exosomes (LEXK562) derived from BCR-ABL positive leukemia K562 cells were isolated and purified by ultracentrifugation. The expression of heat shock protein 70 (HSP70) and BCR-ABL in LEXK562 cells was detected by immunoelectron microscopy and Western blotting. The kinetics of LEXK562-targeted binding to DC was detected by laser scanning confocal microscopy and flow cytometry. The anti-leukemia immune response of LEX and its sensitized DCs was detected by LDH release assay and mouse model in vivo. RESULTS: Similar to other cell-derived exosomes, K562 cell-derived extracellular body LEXK562 was a capsular structure with a diameter of 50-100 nm and expressed K562 cell-specific BCR-ABL and HSP70 molecules. LEXK562 can bind to DC in vitro, reached the peak in 3 ~ 4 h and can stably exist in DC for more than 72 h. The cytotoxic T lymphocyte (CTL) induced by LEXK562-sensitized DCs (DC / LEXK562) can effectively kill K562 target cells. The killing activity of LEXK562 induced by LEXK562 is significantly higher than that of LEXK562-induced CTL [ (68.6 ± 5.7)% vs (22.5 ± 2.9)%, P <0.01]. In vivo experiments further confirmed that the tumorigenicity of L1210 cells inoculated with leukemia L1210-derived extracellular body (LEXL1210) was significantly higher than that of LEXL1210 The tumorigenic rate of mice immunized with primed DCs (DC / LEXL1210) [(54.17 ± 8.33)% vs (16.67 ± 4.18)%, P <0.05]. CONCLUSION: LEX can express leukemia-associated antigen. LEX can bind to DC in vitro, and its sensitized DC can induce stronger anti-leukemia immune effect.