佛波酯诱导K562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 分析佛波酯（TPA）诱导K562细胞分化过程中WT1基因及其四种异构体表达水平及比例变化，探讨WT1基因不同异构体与细胞分化的关系。方法采用TPA诱导K562细胞分化，以硝基四氮唑蓝（NBT）还原实验及细胞免疫表型CD9检测判断细胞分化程度；实时荧光定量RT—PCR技术检测K562细胞被诱导分化过程中总WT1基因表达水平，并计算出WT1（＋／＋）、WT1（＋／-）、WT1（-／＋）、WT1（-／-）四种异构体在细胞分化过程中的比例变化。结果 TPA诱导K562细胞分化过程中NBT阳性率及CD9表达阳性率均有显著增加（P〈0．05）。WT1相对表达水平由分化前（4．67±1．11）×10^-3，降到（1．67±0．45）×10^-3（P〈0．05），随后回升，96h达（4．64±1．53）×10^-1；四种异构体比例变化不-致，WT1（＋／＋）比例由0h的（39．65±19．46）％降至96h的（15．25±7．27）％，而WT1（-／-）异构体由0h的（15．38±11．34）％上升至96h的（37．60±11．90）％，另外两种异构体比例变化无显著性差异。结论 TPA诱导K562细胞分化过程中总WT1表达水平24h内迅速下降，随后回升。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的比例而发挥抑制分化或促进分化的作用。

Expression of WT1 gene and its isomer ratio changes during phorbol ester induced differentiation of K562 cell line

OBJECTIVE: To explore the changes in expression of WT1 gene and ration of its isomers during phorbol ester (TPA) induced differentiation of leukemia cell line K562 by fluorescence quantitative RT-PCR and analysis the relationship between different isomers and hematogenic cell differentiation. METHODS: The degree of cellular maturation were verified by NBT reduction test and immunophenotyping. Expression of WT1 gene was determined by fluorescence quantitative RT-PCR during differentiation of K562 cell line. The relative ratio of the four splicing variants WT1 ( + / + ), WT1 ( + / - ), WT1 ( - / + ), WT1 ( - / - ) were calculated. RESULTS: During the differentiation of K562 cell, the NBT reduction rate and the CD9 positive rate both increased significantly (P < 0. 05). The expression of WT1 gene decreased immediately to (1.67 +/- 0.45) x 10(-3) from (4.67 +/- 1.11) x 10(-3), and then increased again to (4.64 +/- 1.53) x 10(-3) at 96 hours. The ratio of WT1 ( + / + ) was decreased gradually, from 0 hour (39.65 +/- 19.46)% to 96 hour (15.25 +/- 7.27)%. While the ratio of WT1( - / - ) was increased, from 0 hour (15.38 +/- 11.34)%, to 96 hour (37.60 +/- 11.90)%. The other two isomers ratios did not change significantly. CONCLUSION: During the TPA induced differentiation of K562 cell, there are two high expression levels of WT1 gene. Before differentiation, the majority is WT1 ( + / + ), and after differentiation, is WT1 ( - / - ). It indicates that WT1 gene may activate or inhibit cell differentiation by regulating the ratio of its four splicing variants.