达沙替尼对NK细胞的体外扩增、细胞亚群、受体表达及细胞毒功能的调节作用  

Effects of Dasatinib on the Expansion,Subsets,Receptor Expression and Cytotoxic Function of NK Cells in Vitro

作　　者：盛立霞[1] 王佳萍[1] 赖艳丽 吴昊 孙永城[1] 周淼 欧阳桂芳[1] 黄河[2] SHENG Li-Xia;WANG Jia-Ping;LAI Yan-Li;WU Hao;SUN Yong-Cheng;ZHOU Miao;OUYANG Gui-Fang;HUANG He(Department of Hematology,Ningbo First Hospital,Ningbo 315010,Zhejiang Province,China;Bone Marrow Transplantation Center,The First Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310003,Zhejiang Province,China)

机构地区：[1]宁波市第一医院血液科,浙江宁波315010 [2]浙江大学医学院附属第一医院骨髓移植中心,浙江杭州310003

出　　处：《中国实验血液学杂志》2020年第5期1762-1768,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金青年基金项目(81401321);浙江省基础公益研究计划项目(LGF19H080002);宁波市公益类项目(2019C50068)。

摘　　要：目的:探讨达沙替尼对NK细胞的体外扩增作用,以及对NK细胞的亚群、受体表达及细胞毒功能的影响。方法:从健康成人外周血中分离单个核细胞(PBMC),采用添加过IL-2、IL-15的SCGM培养液进行NK细胞的扩增培养,培养体系中添加不同浓度的达沙替尼,细胞计数结合FCM检测分析扩增效率,FCM检测NK细胞表面受体表达以及亚群的分布;采用脱颗粒试验结合CFSE/7AAD杀伤试验检测NK细胞对白血病细胞株K562的细胞毒功能。结果:5-50 nmol/L的达沙替尼能够在体外增加NK细胞的扩增效率,NK细胞的扩增效率在达沙替尼浓度为20 nmol/L时达到峰值。20 nmol/L达沙替尼培养组的NK细胞对K562细胞的细胞毒作用强于对照组。达沙替尼体外扩增的NK细胞中,CD226、NKP46及NKG2D等活化型受体的MFI上调;NKG2A^+CD57^-亚群比例下调,而NKG2A^-CD57^+亚群比例增高,NKG2A^-CD57^+NK细胞对K562细胞的脱颗粒反应强于NKG2A^+CD57^-NK细胞。结论:20 nmol/L的达沙替尼可以增强NK细胞体外扩增的效率,并能够上调扩增的NK细胞表面活化型受体的表达和优先扩增胞毒功能较强的NKG2A^-CD57^+亚群,从而增强NK细胞对白血病细胞株的细胞毒作用。Objective:To investigate the effect of dasatinib on the expansion of NK cells in vitro,as well as the subsets,receptor expression and cytotoxic function of NK cells.Methods:Peripheral blood mononuclear cells(PBMCs)were isolated from healthy adult volunteers and cultured with SCGM added IL-2 and IL-15 for expansion of NK cells.In this culture system,dasatinib of different concentrations were added.Cell counting and phenotyping by flow cytometry were used to evaluate the amplification efficiency of NK cells.FCM was used to detect the expression of receptors on the surface of NK cells and the distribution of subsets.Subsequently,degranulation assay and CFSE/7 AAD based cytotoxicity assay were used to detect the effects of dasatinib on NK cytotoxicity against leukemia cell line K562 cells.Results:The expansion efficiency of NK cells in vitro could be increased by dasatinib at the concentration range of5-50 nmol/L,and the expansion efficiency of NK cells reached the peak at 20 nmol/L of dasatinib.The NK cytotoxicity against K562 cells in dasatinib cultured group at the concentration of 20 nmol/L was significantly higher than that in control group.For the cells cultured by disatinib in vitro,the MFI of CD226,NKP46 and NKG2 D was upregulated;the ratio of NKG2A^+CD57^-subset was down-regulated,while the ratio of NKG2A^-CD57^+subset was upregulated.The degranulation response of NKG2A^-CD57^+NK cells to K562 cells was stronger than that of NKG2A^+CD57^-NK cells.Conclusion:The results shows that appropriate dose of dasatinib(20 nmol/L)can increases the amplification efficiency of NK cells,simultaneously up-regulates the expression of NK activating receptors and increases the NKG2A^-CD57^+subset,which lead to the enhancement of NK cytotoxicty against leukemia cell lines.

关 键 词：达沙替尼 自然杀伤细胞 体外扩增 细胞毒作用 激活型受体 

分 类 号：R733[医药卫生—肿瘤] R329[医药卫生—临床医学]