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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(normalizedWT1expressionlevel),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0,6,12,18,24,48,72h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ATRA作用后开始的18h内,WT1(17AA+)N /WT1N比值逐渐下降,24h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。
Objective To investigate the relationship between the expression of WT1 (17AA+/-) splice variants and the differentiation of NB4 cells. METHODS: Real-time RT PCR was used to detect the expression level of housekeeping genes GAPDH, WT1 and their WT1 (17AA+) splice variants. WT1 expression was calculated using the same sample (WT1 copy number/GAPDH copy number)×104. Defined as WT1N (normalized WT1 expression level), WT1 (17AA+)N was also defined, and flow cytometry was used to detect the change of CD11b surface antigen of NB4 cells. Results The expression level of WT1 gene and its WT1 (17AA+) splicing variants were rapidly decreased as NB4 cells were terminally differentiated from granules. All-trans retinoic acid (ATRA) effect was 0, 6, 12, 18, 24, 48, 72h. The average expression levels of post-WT1N were 191.11, 121.17, 66.72, 43.47, 18.29, 4.04 and 3.79 respectively. The expression levels of WT1(17AA+) N splice variants were 105.12, 46.89, 20.50, 10.38, 8.85, 2.16 and 1.92, respectively. All were negatively correlated with changes in CD11b (r=-0.65, P<0.01; r=-0.77, P<0.01), and it is worth noting that WT1(17AA+)N/WT1N occurred within 18 h after the onset of ATRA. The ratio gradually decreased, and there was no statistical difference between the 24 h and the pre-drug level, indicating that the expression of the WT1 (17AA+) splice variant was mainly decreased in the early stage of NB4 cell differentiation induced by ATRA. Conclusion The high expression of WT1 gene is related to the differentiation arrest of NB4 leukemia cells. WT1 (17AA+) splice variants may play a major role in differentiation arrest.