全反式维A酸诱导的部分分化的白血病细胞具有去分化能力

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)。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。

ATRA-induced partially differentiated leukemia cells retain de-differentiation potential

Objective： To study the de-differentiation capacity of partially differentiated leukemia cells of human acute promyelocytic leukemia cell line NB4 cells induced by all-traMs retinoic acid （ATRA）. 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, and 336 h. The morphology and expression of cell surface differentiation antigen CDllb in treated NB4 cells were monitored by Wright＇s stain and flow cytometry, respectively. Western blot was used to detect the protein levels of PML-RARa, RARa, and PU.1. The phenotype of CDllb cells at different time points were sorted out , and clonal recovery capacity by single cell was assayed in the absence of ATRA. Results： The expression of CD1 lb reached the peak when NB4 cells were exposed to ATRA for 48 h; the percentage of mature neutrophils increased along with the elongation of ATRA exposure, reaching maximum level during 96 h to 120 h after ATRA exposure（P〈0.01）. The expression of PML-RARa declined gradually opposite to the rise in expression of RARer and PU.1. However, a portion of these viable CDllb NB4 cells were still able to re-establish leukemia clones, with restoration to original morphology and expression level of PML-RARa and decrease in expression of CD1 lb. Of note, this de-differentiation ability and clone re-forming rate of NB4 cells decreased with the elongation of ATRA exposure, and the viable progeny of NB4 cells had completely lost the clonal recovery potential after 336 h ATRA exposure. Interestingly, the maturation of NB4 progeny cells to neutrophil after ATRA exposure was negatively correlated with the loss of clone recovery capacity （r=-0.905, P〈0.01 ）. Conclusions： The NB4 cells respond to differentiation-induction of ATRA in a step-wise manner, and the partially differentiated leukemia cells are still able to regain the characteristic of unlimited proliferation by de-differentiation when ATRA is removed.