Daxx抑制K562细胞向巨核细胞分化

目的:观察过表达死亡结构域相关蛋白(Daxx)对K562细胞活力和向巨核细胞分化的影响。方法:建立稳定过表达Daxx的K562细胞,荧光显微镜观察、实时荧光定量PCR和Western blot检测Daxx的过表达效果,CCK-8法检测过表达后细胞活力的变化;佛波酯(PMA)诱导K562细胞向巨核细胞系分化,Western blot检测在K562细胞向巨核细胞分化过程中Daxx和p-ERK的表达变化,流式细胞术检测CD41和CD61的表达变化;PMA处理过表达Daxx的K562细胞,NBT还原实验检测细胞分化情况,流式细胞术检测过表达Daxx后CD41和CD61的表达变化,Western blot检测p-ERK的蛋白水平。结果:建立了稳定的过表达Daxx的K562细胞,过表达Daxx抑制K562细胞的活力。PMA诱导K562细胞向巨核细胞分化,CD41和CD61表达水平增高,同时p-ERK的蛋白水平升高,Daxx表达水平下降。过表达Daxx可以抑制K562细胞向巨核细胞分化,CD41和CD61表达降低,同时p-ERK的蛋白水平降低。结论:过表达Daxx可以抑制K562细胞生长及向巨核细胞分化,同时抑制ERK的磷酸化。

Role of Daxx in inhibiting megakaryocytic differentiation of K562 cells

AIM: To examine the effects of death domain-associated protein (Daxx) overexpression on the viability and megakaryocytic differentiation of K562 cells. METHODS: Daxx overexpression in the K562 cells was established. The expression of Daxx was detected by fluorescence microscopy, fluorescence quantitative real-time PCR and Western blot after transfection. CCK-8 assay was used to detect the cell viability after overexpression of Daxx. The expression of CD41 and CD61 in phorbol 12-myristate 13-acetate (PMA) induced K562 cells was detected by flow cytometry. The protein levels of Daxx and p-ERK were determined by Western blot. Nitroblue tetrazolium (NBT)-reducing test was used to assess leukemia cell differentiation in Daxx-overexpressing K562 cells and control cells. The expression of CD41 and CD61 induced by PMA in Daxx-overexpressing K562 cells was analyzed by flow cytometry. The protein levels of Daxx and p-ERK were also examined by Western blot. RESULTS: The stable overexpression of Daxx in the K562 cells was established. The viability was reduced in Daxx-overexpressing K562 cells. The expression of CD41 and CD61 was significantly increased after PMA induction in the K562 cells (P < 0.01). The protein expression of Daxx was reduced, but the protein level of p-ERK was increased. The expression of CD41 and CD61 was reduced after PMA induction in Daxx-overexpressing K562 cells (P < 0.01). The protein level of p-ERK was also reduced. CONCLUSION: Daxx overexpression inhibits the growth, megakaryocytic differentiation and production of p-ERK in the K562 cells.