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目的:观察人急性早幼粒细胞白血病(acute promyelocytic leukemia,APL)细胞系NB4细胞株经全反式维A酸(all-trans-retinoic acid,ATRA)诱导分化作用后,部分分化的白血病细胞的去分化能力。方法 :采用1μmol/L的ATRA处理NB4细胞48 h、72 h、96 h、120 h、168 h、216 h、288 h、336 h后,并通过瑞氏染色观察细胞形态学,流式细胞术检测CD11b的表达,用单克隆形成实验分别检测分化细胞的核型及撤药后各时间点单个CD11b+细胞的克隆形成能力,用蛋白印迹法观察细胞PML/RARα、RARα及PU.1等蛋白的表达变化。结果:NB4细胞CD11b的表达水平在ATRA处理48 h后达到高峰,之后稳定表达;ATRA处理后,NB4细胞中成熟中性粒细胞的比例也随处理时间的延长而增加,ATRA处理120 h比处理96 h,成熟中性粒细胞的比例有明显的增加(P<0.01),之后比例逐步增加至平台期;且NB4细胞中PML-RARα的表达水平逐渐下降,伴随RARα及PU.1的表达上升。在撤去ATRA后,部分CD11b+的NB4活细胞能够重建白血病细胞克隆,形态上回复到诱导分化前的原始细胞形态,CD11b的表达也降低,PML-RARα的表达水平也恢复到处理前水平。尤其是NB4细胞的去分化能力及克隆形成率随着ATRA处理时间的增加而降低,直至ATRA处理至336 h,NB4子代细胞才完全丧失了克隆形成能力。ATRA处理后,NB4子代细胞的克隆形成能力与其成熟中性粒细胞的比例呈负相关(r=-0.905,P<0.01)。结论:人类APL系NB4细胞经ATRA诱导后发生不同程度的分化,在撤去ATRA后,处于部分分化阶段的白血病细胞仍能通过“去分化”而重新获得无限增殖的特性。
OBJECTIVE: To observe the effect of allotrans-retinoic acid (ATRA) on differentiation of NB4 cell line from human acute promyelocytic leukemia (APL) To differentiation ability. Methods: NB4 cells were treated with 1 μmol / L ATRA for 48 h, 72 h, 96 h, 120 h, 168 h, 216 h, 288 h, 336 h. The morphological changes of NB4 cells were observed by Wright’s stain. Flow cytometry The expression of CD11b was detected. The karyotypes of differentiated cells and the clonogenic capacity of individual CD11b + cells at various time points were detected by monoclonal formation assay. The expressions of PML / RARα, RARα and PU.1 proteins were detected by Western blotting Change of expression Results: The expression level of CD11b in NB4 cells peaked at 48 h after ATRA treatment and then stably expressed. After ATRA treatment, the proportion of mature neutrophils in NB4 cells also increased with the prolongation of treatment time. After ATRA treatment for 120 h, 96 h, the proportion of mature neutrophils was significantly increased (P <0.01), and then gradually increased to the plateau phase; and the expression of PML-RARα in NB4 cells gradually decreased, accompanied by an increase in the expression of RARα and PU.1 . After the withdrawal of ATRA, some CD11b + NB4 live cells were able to reconstitute leukemic cell clones, morphologically back to the original cell morphology before differentiation, the expression of CD11b was also reduced, and the expression level of PML-RARα was restored to its pre-treatment level. In particular, the ability of dedifferentiation and clonogenicity of NB4 cells decreased with the increase of ATRA treatment time. Until the passage of ATRA to 336 h, the NB4 progeny cells completely lost their clonogenic capacity. After ATRA treatment, the clonogenic capacity of NB4 progeny cells was negatively correlated with their proportion of mature neutrophils (r = -0.905, P <0.01). CONCLUSION: Human APL line NB4 cells differentiate to different extents after induction by ATRA. After the ATRA withdrawal, leukemic cells in the partially differentiated stage can still regain their immortalized characteristics through “dedifferentiation”.