地西他滨增强allo-NK细胞杀伤白血病干细胞敏感性研究

Decitabine Enhances the Sensitivity of Leukemia Stem Cell to Allo-NK Cell-Mediated Killing

目的:探讨地西他滨增强allo-NK细胞杀伤白血病干细胞(LSC)的作用及其机制。方法:应用免疫磁珠法从KG1a细胞中分选LSC。从健康供者的外周血分离纯化同种异体反应性自然杀伤(allo-reactive natural killer,a1-Io-NK)细胞;LDH法检测allo-NK细胞对LSC的杀伤作用,流式细胞术检测allo-NK细胞诱导LSC凋亡及LSC表面NKG2D配体MICA/B和ULBP1-3的表达。结果:地西他滨10μmol/L处理LSC 24 h后,allo-NK细胞对LSC的杀伤率明显增高,在效靶比为5:1、10:1、20:1时分别为(60.52%±3.52%vs 22.08%±2.07%、73.93%±2.33%vs 28.99%±3.13%、83.08%±1.32%vs 36.44%±2.40%),差异有统计学意义(P<0.05);地西他滨10μmol/L处理LSC 24 h后,在效靶比为10:1时,allo-NK细胞诱导LSC的凋亡率为7.84%±0.34%,明显高于LSC未处理组(3.33%±0.64%),差异有统计学意义(P<0.05);地西他滨10μmol/L处理LSC 24 h后,LSC表面NKG2D配体(MICA/B、ULBP1、ULBP2、ULBP3)的表达上调,高于未处理组,差异有统计学意义(P<0.05)。结论:地西他滨可能通过上调NKG2D配体增强allo-NK细胞对LSC的杀伤作用。

Objective;To investigate the allo-NK cell-mediated killing effect enhanced by decitabine on leukemia stem cells（LSC） and the underlying mechanisms.Methods;LSC were separated from KG1 a cells by using immunomagnetic beads.Allo-NK cells were isolated and purified from PBMC of healthy donors.Cytotoxicity of alloNK cells against LSC were measured by LDH releasing assay.The apoptosis induced by allo-NK cells in LSC and the expressions of NKG2 D ligands including MICA/B and ULBP1-3 on LSC were detected by flow cytometry.Results：The killing rate of allo-NK cells to LSC treated with 10 μmol/L decitabine for 24 hours was significant higher than that to LSC without treatment 60.52%±3.52%vs22.08%±2.07%,73.93%±2.33%vs 28.99%±3.13%,83.08%± 1.32%vs 36.44%±2.40%,respectively） at the effector-target ratios of 5：1,10：1,20：1（P〈 0.05）.At the effector-target ratio of 10：1,decitabine significantly enhanced the apoptosis of LSC induced by allo-NK cells（7.84%±0.34%vs 3.33%±0.64%）（P〈0.05）.The expressions of NKG2 D ligands（MICA/B,ULBP1,ULBP2,ULBP3） on LSC treated with decitabine 10 μmol/L for 24 hours were significantly increased（P〈 0.05）.Conclusion;Decitabine may enhance the allo-NK cell-mediated killing effects on LSC by up-regulation of the expressions of NKG2 D ligands on LSC.