白藜芦醇对髓系白血病干细胞增殖和凋亡的影响及其与allo-NK细胞的联合杀伤作用

目的探讨白藜芦醇(RES)对髓系白血病干细胞(CD34+CD38-KG1a细胞,以下简写为KG1a细胞)增殖和凋亡的影响,并初步探讨RES增强allo-NK细胞对KG1a细胞杀伤作用的机制。方法免疫磁珠分选法分选人外周血单个核细胞(PBMCs)中的CD16+CD56+CD3-NK细胞(以下简写为NK细胞)。流式细胞术检测细胞表面分子CD34、CD38、CD3、CD15和CD56。MTT法、甲基纤维素克隆形成实验检测RES对KG1a细胞增殖抑制作用。DAPI染色法和流式细胞术检测RES对KG1a细胞凋亡的影响。使用IC50的RES作用KG1a细胞24 h后清洗离心获得RES/KG1a细胞。LDH释放法检测NK细胞对KG1a细胞和RES/KG1a 2种细胞的杀伤能力。流式细胞术检测KG1a细胞和RES/KG1a细胞的表面配体ULBP1、ULBP2、ULBP3、MICA和MICB。结果免疫磁珠分选后,外周血单个核细胞中的NK细胞比例为(90.5±3.2)%;KG1a细胞纯度为(91.2±3.9)%。RES对KG1a细胞的增殖抑制作用具有时间和剂量依赖性,24 h的IC50=24.0μmol·L-1;48 h的IC50=13.7μmol·L-1。RES抑制KG1a细胞的克隆形成能力并诱导细胞凋亡,具有剂量依赖性(P<0.05)。NK细胞对RES/KG1a细胞的杀伤能力比其对KG1a细胞的杀伤能力强(P<0.05)。与KG1a细胞比较,RES/KG1a细胞表面配体ULBP1、ULBP2、ULBP3表达明显上调(P<0.05),MICA和MICB变化不明显。结论 RES能够抑制KG1a细胞增值抑制并诱导细胞凋亡,联合NK细胞对KG1a细胞具有协同杀伤作用,这可能与KG1a细胞表面的ULBP1、ULBP2、ULBP3表达上调相关。

Effect of resveratrol on proliferation and apoptosis of myeloid leukemia stem progenitor cells and its synergistic cytolysis with allo-NK cells

Objective To investigate the effect of resveratrol（RES） on the proliferation and apoptosis in myeloid leukemia stem progenitor cells（CD34 ＋ CD38-KG1a）,and to explore the underlying mechanism of RES enhancing the cytotoxicity of allo-NK cells against the KG1a.Methods CD16 ＋ CD56 ＋ CD3-NK cells were sorted from peripheral blood mononuclear cells（PBMCs） by immunomagnetic beads.Flow cytometry was used to detect the surface molecules（CD34,CD38,CD3,CD15 and CD56） in the cells.MTT assay and methylcellulose colony formation assay were applied to examine the inhibitory effect of RES on the proliferation of KG1a cells.DAPI staining method and flow cytometry were used to detect the influence of RES on the apoptosis of KG1a cells.The KG1a cells was treated with RES（IC50） for 24 h and RES/KG1a cells was available.Cytolysis of NK cells to 2 KG1a cells and RES/ KG1a cells was analyzed by LDH release assay.Flow cytometry was used to detect the surface ligands ULBP1,ULBP2,ULBP3,MICA and MICB in KG1a cells and RES/ KG1a cells.Results The proportion of NK cells in PBMCs was（90.5±3.2）% after the sorting.The purity of KG1a cells was（91.2±3.9）% in KG1a cell lines.The inhibitory effect of RES on the proliferation in KG1a cells was in a time and dose dependent manner（24 h IC50=24.0 μmol·L-1; 48 h IC50=13.7 μmol·L-1）.The colony ability of KG1a cells was inhibited and apoptosis induced by RES in a dose dependent manner（P 〈0.05）.The cytolysis effect induced by NK cells was stronger in RES/KG1a than that in KG1a（P 〈0.05）.Compared with KG1 a,the surface ligands ULBP1,ULBP2,and ULBP3 were significantly increased（P0.05） in RES/KG1a cells,while MICA and MICB had no obvious change.Conclusion RES can inhibit the proliferation and induce the apoptosis of KG1 a,with synergistic cytotoxicity with allo-NK cells,which may be related to the upregulation of surface ligands ULBP1,ULBP2 and ULBP3.