维甲酸诱导HL-60细胞分化过程中WT1基因表达及其异构体比例变化的研究

目的 探讨全反式维甲酸(ATRA)诱导HL-60细胞分化过程中WT1基因及其异构体表达水平及比例变化.方法 采用ATRA诱导HL-60细胞分化,以硝基四氮唑蓝(NBT)还原实验及细胞免疫标记CD11b检测判断细胞分化程度;即时荧光定量RT-PCR方法检测HL-60细胞在诱导分化过程中总WT1、WT1(17AA+)及WT1(KTS+)的表达,并计算出WT1(+/+)、WT1(+/-)、WT1(-/+)、WT1(-/-)四种异构体的比例.结果 ATRA诱导HL-60细胞分化过程中NBT阳性率及CD11b表达阳性率与诱导分化前(0 h)相比均显著增加(P<0.05,P<0.001).随着细胞分化,WT1表达水平由0 h的(4.17±2.21)× 10~(-3)降低至96 h的(7.53±2.30)×10~(-4);17AA+和KTS+两类异构体的比例也逐渐下降,17AA+异构体比例由0 h的0.60±0.05降到96 h的0.42±0.08(P<0.05).KTS+异构体比例由0.53±0.08降至96 h的0.41±0.04(P<0.05);四种异构体的比例变化表现不一致,WT1(+/+)比例由0 h的0.32±0.06降至96 h的0.17±0.03,而WT1(-/-)比例由0 h的0.19±0.04升至96 h的0.34±0.05,另外两类异构体比例变化差异无统计学意义.结论 ATRA诱导HL-60细胞分化过程中总WT1表达水平逐渐降低,分化前异构体以WT1(+/+)为主,而分化后以WT1(-/-)为主,提示WT1基因可能通过调节四种异构体比例而发挥抑制或促进分化的作用.

Alteration of the expression ratio between WT1 gene and its isomers during all-trans retinoic acid induced differentiation of HL-60 cells

Objective To explore expression ratio alteration between WT1 gene and its isomers during differentiation of leukemia cell line HL-60 induced by all-trans retinoic acid ( ATRA ) and the relationship existed between them.Methods The degree of cellular maturation was verified by NBT reduction test and immunophenotyping.Expression of unspliced WT1, WT1 ( 17AA + ) and WT1 ( KTS + )were determined by real-time fluorescence quantitative RT-PCR during the induced differentiation of HL-60 cells.The relative ratio of four splicing variants WT1 ( +/+ ), WT1 ( +/- ), WT1 ( -/+ ), WT1( -/- ) were analyzed.Results During the induced differentiation of HL-60 cells, NBT reduction rate and CD11b positive rate increased significantly(P < 0.05 and P < 0.001, respectively).The expression of WT1 gene decreased from (4.17±2.21 ) × 10 -3 at 0 hour to (7.53 ±2.30) × 10 -4 at the 96th hour.The ratio of 17AA +decreased from 0.60±0.05 at 0 hour to 0.42±0.08 at the 96th hour.The ratio of KTS +decreased from 0.53 ± 0.08 at 0 hour to 0.41 ± 0.04 at the 96th hour.The ratio of WT1 ( + / + )decreased gradually from 0.32±0.06 at 0 hour to 0.17±0.03 at the 96th hour.Change of ratios of other two isomers not significant.Conclusions During the induced differentiation of HL-60 cells, WT1 gene expression decreases gradually.The phenotype of the majority of uninduced HL-60 cells is WT1 ( +/+ ),in contrast to WT1 ( - / - ) phenotype after the induction of cell differentiation, indicating that WT1 gene may participate in the regulation of hematopoietic cell differentiation through modulation of the expression ratios of its four spliced variants.