摘要
目的探讨CD34^+CD38^-KG1a白血病干细胞抵抗同种异体自然杀伤(NK)细胞杀伤作用的机制。方法采用免疫磁珠法(MACS)分选CD34^+CD38^-KG1a细胞和4例健康个体外周血中NK细胞,并采用流式细胞术检测纯度;乳酸脱氢酶(LDH)释放法检测NK细胞在不同效靶比(5∶1,10∶1,20∶1)下对K562和CD34^+CD38^-KG1a细胞的杀伤作用;聚合酶链式反应-序列特异性引物法分析NK细胞KIR和CD34^+CD38^-KG1a细胞HLA-Ⅰ基因分型;流式细胞术检测K562和CD34^+CD38^-KG1a细胞表面主要组织相容性复合体(MHC)-I类链相关分子A/B、人巨细胞病毒UL16结合蛋白(ULBP)1~3和人类白细胞抗原(HLA)-Ⅰ类分子的表达。结果分选的CD34^+CD38^--KG1a细胞和NK细胞纯度分别为(94.25±2.16)%、(91.70±2.05)%。NK细胞在各效靶比下对CD34^+CD38^-KG1a细胞的杀伤率均低于K562细胞(P<0.05)。4例健康个体NK细胞KIR基因型为KIR2DL1、KIR2DL3、KIR3DL1和KIR3DL2,CD34^+CD38^-KG1a细胞HLA-Ⅰ基因型为A30、30,B51、78,Cw4、16。K562细胞高表达NKG2D配体,CD34^+CD38^-KG1a细胞几乎不表达NKG2D配体,CD34^+CD38^-KG1a细胞中NKG2D配体表达率显著低于K562细胞(P<0.05);K562细胞中几乎不表达HLA-Ⅰ,CD34^+CD38^-KG1a细胞高表达HLA-Ⅰ,CD34^+CD38^-KG1a细胞中HLA-Ⅰ的表达率显著高于K562细胞(P<0.05)。结论CD34^+CD38^-KG1a细胞明显抵抗同种异体NK细胞的杀伤作用,其机制可能与高表达HLA-Ⅰ分子和低表达NK2D配体有关。
Objective To explore the mechanism of CD34^+CD38^-KG1a leukemia stem cells against killing effect of nature killer (NK)cells. Methods The CD34^+CD38^-KG1a cells and NK cells of 4 healthy individuals were isolated by magnetic cell sorting (MACS) and the purity were detected by flow cytometry.The killing effects of NK cells on K562 and CD34^+CD38^-KG1a cells at different effector-to-target cell ratios(5∶1,10∶1,20∶1) were detected by lactate dehydrogenase (LDH) release assay.KIR genotyping of NK cells and HLA-I genotyping of CD34^+CD38^-KG1a cells were analyzed by polymerase chain reaction-sequence specific primers.The expression of major histocompatibility complex(MHC) class I chain-related molecules A and B(MICA/B),human cytomegalovirus glycoprotein UL16 binding proteins(ULBP)1-3 and human leukocyte antigen(HLA)-I molecules on the surface of K562 and CD34^+CD38^-KG1a cells were detected by flow cytometry. Results The purity of CD34^+CD38^-KG1a cells and NK cells were (94.25+2.16)% and (91.70+2.05)% respectively.The killing rate of NK cells to CD34^+CD38^- KG1a cells was lower than that to the K562 cells under all effector-to-target cell ratios ( P <0.05).The KIR genotypes of NK cells in 4 healthy individuals were KIR2DL1,KIR2DL3,KIR3DL1 and KIR3DL2;the HLA-I genotypes of CD34^+CD38^-KG1a cells were A30,30;B51,78;Cw4,16.NKG2D ligand was highly expressed in K562 cells,and NKG2D ligand was almost not expressed in CD34^+CD38^-KG1a cells;the expression rate of NKG2D ligand in CD34^+CD38^-KG1a cells was significantly lower than that in the K562 cells ( P <0.05).The HLA-I was almost not expressed in K562 cells,but it was highly expressed in CD34^+CD38^-KG1a cells;the expression rate of HLA-I in CD34^+CD38^-KG1a cells was significantly higher than that in the K562 cells( P <0.05). Conclusion CD34^+CD38^-KG1a cells can significantly resistant the killing of allogeneic NK cells,which may be related to the high expression of HLA-I and the low expression of NK2D ligands.
作者
王国征
吴远彬
郭坤元
代喜平
WANG Guo-zheng;WU Yuan-bin;GUO Kun-yuan;DAI Xi-ping(Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510120,Guangdong Province,China)
出处
《新乡医学院学报》
CAS
2019年第9期828-832,共5页
Journal of Xinxiang Medical University
基金
广东省科技计划项目(编号:2017KT1747)