探讨健康供者（HD）与急性B淋巴细胞白血病患者（B-ALL）骨髓单个核细胞中的自然杀伤（NK）细胞，以及在体外活化扩增产生的NK细胞生物学表型和细胞毒作用的异同。

收集HD和B-ALL患者的骨髓血，经过密度梯度离心富集单个核细胞，采用流式细胞术分析二者的驻留型NK细胞（HD-NK，BALL-NK）的表型；基于课题组建立的"3ILs [白细胞介素-2 （IL-2），IL-15，IL-18]"体外高效活化扩增体系，采用细胞计数、流式细胞分析和体外共培养杀伤实验，比较二者培养14 d后获得的扩增型eHD-NK细胞和eBALL-NK细胞的表型和细胞毒作用。组间比较采用独立样本t检验；HD-NK、eHD-NK、BALL-NK、eBALL-NK四组间的比较采用两因素重复测量方差分析，两两比较采用Bonferroni事后检验。

与HD组相比，B-ALL患者CD3^-CD56⁺总NK细胞比例[（4.21 ± 0.92）%比（14.25 ± 1.15）%]、CD16⁺ NK细胞[（46.82 ± 2.96）%比（67.87 ± 2.12）%]、NKG2D⁺ NK细胞[（22.45 ± 2.12）%比（50.82 ± 5.65）%]和NKp46⁺ NK细胞比例[（7.66 ± 1.73）%比（17.27 ± 1.75）%]均降低（P均< 0.05）。经过体外活化扩增的eHD-NK组和eBALL-NK组中的总NK细胞比例均提升，与eBALL-NK组比较，eHD-NK组的总NK细胞比例[（61.52 ± 3.18）%比（24.63 ± 2.07）%]、NKG2D⁺ NK细胞[（80.63 ± 2.03）%比（57.83 ± 8.55）%]、CD25⁺ NK细胞[（37.45 ± 3.21）%比（20.90 ± 5.15）%]和NKp46⁺NK细胞比例[（27.23 ± 2.30）%比（9.51 ± 0.98）%]均升高（P均< 0.05）。细胞活力分析显示，HD组的总NK细胞体外活化扩增效率高于B-ALL组。与eHD-NK组比较，eBALL-NK组的Annexin Ⅴ⁺ 7-AAD^- [（4.50 ± 0.35）%比（2.72 ± 0.43）%]和Annexin Ⅴ⁺细胞凋亡比例[ （5.13 ± 0.62）%比（3.29 ± 0.58）%]均升高，两组间细胞周期分布差异无统计学意义。体外杀伤共培养实验显示，eHD-NK对肿瘤细胞系Nalm6、U937的杀伤能力高于eBALL-NK组。

相较于HD，B-ALL患者骨髓血来源的驻留型和活化型NK细胞比例、活化亚群含量、体外活化能力、细胞活力和细胞毒等方面均较低，这一研究为后续探究致病机制和临床治疗提供有益参考。

To explore the similarities and differences in the biological phenotypes and cytotoxicity of resident and expanded natural killer cells derived from mononuclear cells of human bone marrow between healthy donors (HD) and patients with B-cell acute lymphoblastic leukemia (B-ALL) .

Bone marrow blood of HD and B-ALL was collected, and mononuclear cells were enriched by density gradient centrifugation. Flow cytometry was used to analyze the phenotypes of resident NK cells (HD-NK and BALL-NK) in the two types of blood. Then, with the aid of our well-established 14-day "3ILs"-based (short for IL-2, IL-15, IL-18) culture system for in vitro high-efficient activation and expansion of NK cells, we conducted cell counting, flow cytometry analysis and in vitro co-culture killing tests to compare the phenotypes and cytotoxicity of expanded HD-NK cells (eHD-NK) and expanded BALL-NK cells (eBALL-NK) in bone marrow blood. Comparisons between two groups were performed using independent samples t-test. Comparisons among the four groups (HD-NK, eHD-NK, BALL-NK, and eBALL-NK) were conducted using two-way repeated measures ANOVA, followed by Bonferroni's post hoc test for pairwise comparisons.

Compared with the healthy donor (HD) group, the proportion of total CD3^-CD56⁺ NK cells in the non-activated, expanded resident NK cell population from patients with B-cell acute lymphoblastic leukemia (B-ALL) was significantly lower [ (4.21 ± 0.92) % vs (14.25 ± 1.15) %]. In addition, the frequencies of activated NK cell subsets were reduced, including CD16⁺ NK cells [ (46.82 ± 2.96) % vs (67.87 ± 2.12) %], NKG2D⁺ NK cells [ (22.45 ± 2.12) %vs (50.82 ± 5.65) %], and NKp46⁺ NK cells [ (7.66 ± 1.73) % vs (17.27 ± 1.75) %]. Following in vitro activation and expansion, the proportion of total NK cells was significantly increased in both the expanded HD-NK (eHD-NK) group and the expanded B-ALL-derived NK (eBALL-NK) group [ (61.52 ± 3.18) % vs (14.25 ± 1.15) %]; (24.63 ± 2.07) % vs (4.21 ± 0.92) %]. Moreover, the eHD-NK group exhibited higher proportions of total NK cells [ (61.52 ± 3.18) % vs (24.63 ± 2.07) %] and multiple activated NK cell subsets than the eBALL-NK group, including NKG2D⁺ NK cells [ (80.63 ± 2.03) % vs (57.83 ± 8.55) %], CD25⁺ NK cells [ (37.45 ± 3.21) %vs (20.90 ± 5.15) %], and NKp46⁺ NK cells [ (27.23 ± 2.30) % vs (9.51 ± 0.98) %]. Cell viability analysis demonstrated that the efficiency of in vitro activation and expansion of total NK cells was higher in the HD group than in the B-ALL group. Compared with the eHD-NK group, the eBALL-NK group showed significantly higher proportions of apoptotic cells, including Annexin V⁺7-AAD- cells [ (4.50 ± 0.35) % vs (2.72 ± 0.43) %] and Annexin V⁺ cells [ (5.13 ± 0.62) % vs (3.29 ± 0.58) %], while no significant differences in cell cycle distribution were observed between the two groups. In vitro cytotoxicity co-culture assays revealed that eHD-NK cells exhibited stronger cytotoxic activity against the tumor cell lines Nalm6 and U937 compared with eBALL-NK cells.

Compared with HDs, bone marrow–derived resident and activated NK cells from B-ALL patients exhibited reduced proportions, diminished activated subpopulation content, impaired in vitro activation capacity, decreased cellular viability, and attenuated cytotoxicity. This study provides valuable reference for subsequent investigations into the underlying pathogenic mechanisms and the development of clinical therapeutic strategies.