NB4细胞诱导分化过程中WT1(17AA+/-)剪接变异体表达的研究

目的探讨WT1(17AA /-)剪接变异体表达的改变与NB4细胞诱导分化的关系.方法建立了实时定量RT-PCR方法检测看家基因GAPDH、WT1基因及其WT1(17AA )剪接变异体的表达水平,以同一份标本(WT1拷贝数/GAPDH拷贝数)×104来计算WT1表达水平,定义为WT1N(normalized WT1 expressionlevel),同样定义WT1(17AA )N,并用流式细胞仪检测NB4细胞表面抗原CD11b的变化.结果随着NB4细胞向粒系终末分化WT1基因及其WT1(17AA )剪接变异体的表达水平迅速下降,全反式维甲酸(ATRA)作用0,6,12,18,24,48,72 h后WT1N的平均表达水平分别为191.11,121.17,66.72,43.47,18.29,4.04和3.79,WT1(17AA )N剪接变异体的表达水平分别为105.12,46.89,20.50,10.38,8.85,2.16和1.92,两者均与CD11b的变化呈负相关(r=-0.65,P＜0.01;r=-0.77,P＜0.01),而且值得注意的是在AT-RA作用后开始的18 h内,WT1(17AA )N/WT1N比值逐渐下降,24 h后与药物作用前水平无统计学差异,表明在ATRA诱导NB4细胞分化早期以WT1(17AA )剪接变异体表达下降为主.结论WTl基因高表达与NB4白血病细胞分化阻滞有关,WT1(17AA )剪接变异体可能在分化阻滞中起主要作用.

Detection of WT1 gene isoform expression during induction of differentiation of NB4 cell line

Objective: To investigate the relation between WT1 isoform expression and differentiation of NB4 leukemic cells. Methods: Real-time quantitative retroverse polymerase chain reaction (RQ-RT-PCR) method was established for detecting WT1,WT1(17AA+) isoform and GAPDH expression levels in NB4 leukemic cells induced by retinoic acid (ATRA) by LightCycler. Normalized WT1 expression level (WT1 N ) was determined as a ratio between WT1 and GAPDH times 10 4,so was the WT1(17AA+) isoform, named as WT1(17AA+) N. The expression of CD11b was simultaneously detected with a Becton Dickinson FACSCalibur flow cytometry. Results: WT1 and WT1(17AA+) expression rapidly decreased during the dfferentiation of NB4 cells induced by ATRA. The WT1 N level was 191.11, 121.17, 66.72, 43.47,18.29,4.04 and 3.79 respectively, prior to and at 6, 12, 18,24,48 and 72 hours after exposure to ATRA; and they were in accordance with the dynamic changes of CD11b positive rates( r =-0.65, P <0.01); The WT1(17AA+) N level was 105.12,46.89,20.50,10.38,8.85,2.16 and 1.92,respectively, at the above time points, and they were also in accordance with the CD11b changes ( r =-0.77, P <0.01); Another novel results was that the ratio of WT1(17AA+) N and WT1 N (WT1(17AA+) N/ WT1 N) were significantly decreased during the first 18 hours after exposure to ATRA and re-increased 24 hours later after exposure to ATRA, indicating that during the earlier stage of NB4 cell differentiation, WT1(17AA+) isoform expression significantly decreased. Conclusions: these data suggest that the abnormally high expression of WT1 and WT1(17AA+) isoform were associated with the block of cell differentiation. Of the four main isoforms WT1(17AA+) may dominantly contribute to blocking cell differention.