【摘要】 目的建立稳定抑制水通道蛋白1(AQP1)表达的K562细胞株,探讨AQP1在维甲酸(RA)诱导红白血病细胞红系分化中的作用。方法 RA处理K562细胞,通过real-time PCR法检测γ-珠蛋白表达和分光光度法检测血红蛋白的含量研究RA对K562细胞红系分化的影响。Real-time PCR法、蛋白质印迹法检测RA诱导后K562细胞AQP1转录和蛋白表达水平的变化。设计特异AQP1 shRNA序列,构建pSUPER-retro-puro重组质粒转染K562细胞,筛选建立稳定抑制AQP1基因表达的K562细胞株(K562-shAQP1);通过比较K562-shAQP1细胞株与对照组细胞在维甲酸诱导后γ-珠蛋白和血红蛋白的表达,研究AQP1在维甲酸诱导K562细胞红系分化中的作用。结果 RA处理K562细胞后红系分化指标γ-珠蛋白和血红蛋白的表达均明显增加,同时AQP1 mRNA及蛋白表达随RA作用时间显著增加(P<0.01)。pSUPER-retro-puro-shAQP1干扰载体转染K562细胞后AQP1基因在转录和蛋白水平上的表达均被抑制,差异有统计学意义(P<0.01);K562-shAQP1细胞与对照K562细胞相比,在RA诱导后γ-珠蛋白和血红蛋白表达受到不同程度的抑制,差异有统计学意义(P<0.01)。结论 RA诱导K562细胞红系分化后AQP1表达显著增加,而抑制AQP1基因的表达可部分阻断RA诱导红系分化的作用,说明AQP1在RA诱导K562细胞红系分化过程中发挥重要作用。更多还原

【Abstract】 Objective To explore the role of aquaporin-1(AQP1) gene in erythroid differentiation of erythroleukemia K562 cells induced by retinoic acid(RA).Methods K562 cells were cultured in the presence of 1 μmol/L RA for varying lengths of time,and γ-globin mRNA expression and hemoglobin content in the cells were detected by real-time PCR(RT-PCR) and ultraviolet spectrophotometry,respectively,to evaluate the erythroid differentiation of K562 cells.RT-PCR and Western blotting were used to examine AQP1 expression in the cells following RA treatment.A retroviral expression vector of AQP1 small interfering RNA(pSUPER-retro-puro-shAQP1) was constructed and transfected into K562 cells to establish a K562 cell line with stable AQP1 down-regulation(K562-shAQP1),in which the changes in γ-globin and hemoglobin expressions after RA treatment were detected.Results RA treatment significantly increased γ-globin and hemoglobin expressions in K562 cells(P<0.01),causing also significantly enhanced AQP1 mRNA and protein expressions over time(P<0.01).Transfection with the recombinant plasmids pSuper-retro-puro-shAQP1 resulted in stable AQP1 suppression in K562 cells(P<0.01),which showed markedly reduced γ-globin and hemoglobin expressions after RA induction as compared to the control K562 cells(P<0.01).Conclusions K562 cells show a significant increase of AQP1 expression after RA-induced erythroid differentiation,and suppression of AQP1 expression can partially block the effect of RA,suggesting the important role of AQP1 in RA-induced erythroid differentiation of K562 cells.更多还原