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目的探讨WT1基因异构体对急性早幼粒细胞白血病细胞株NB4分化的影响及其分子机制。方法用电穿孔法将携带WT1基因异构体WTA(-17AA/-KTS)全长cDNA的重组真核表达载体及其对照质粒pCB6+转导NB4细胞,建立WT1基因异构体表达比例改变的单克隆细胞株。通过形态学观察,细胞周期动力学检测,NBT还原试验,细胞表面分化抗原CD11b的检测了解外源性WT1基因异构体WTA在全反式维甲酸(ATRA)诱导下对NB4细胞分化的影响。用RT-PCR检测细胞中PML/RARα、p21、c-myc基因表达的改变。结果ATRA处理细胞48h,对照组(转染空载体的NB4/CMV和未转导WTA的NB4细胞)细胞分化明显,NB4/WTA细胞只出现部分分化。流式细胞术检测NB4/WTA细胞CD11b相对荧光强度(4.63±1.63)低于对照组(8.15±1.84),ATRA作用24h后CD11b相对荧光强度(31.42±5.65)与对照组(71.95±9.36)相比差异更为明显。NB4/WTA细胞NBT还原能力较对照组下降,经ATRA处理后两组细胞之间的NBT还原能力的差异更加明显。RT-PCR提示NB4/WTA细胞PML/RARα、p21、c-myc基因的表达较对照组升高。结论增加外源性WT1基因异构体WTA的表达从而将WT1基因异构体表达的比例由+17AA/+KTS优势型转变为-17AA/-KTS优势型能部分抑制ATRA对NB4细胞的诱导分化作用,这可能与PML/RARα、p21、c-myc基因表达上调有关。
Objective To investigate the effect of WT1 isoforms on the differentiation of acute promyelocytic leukemia NB4 cells and its molecular mechanism. Methods The recombinant eukaryotic expression vector carrying the WT1 gene isoform WTA (-17AA / -KTS) full-length cDNA and its control plasmid pCB6 + were transduced into NB4 cells by electroporation. The expression of WT1 gene isoform Clone cell lines. The morphological observation, cell cycle kinetic assay, NBT reduction assay and cell surface differentiation antigen CD11b assay were used to investigate the effect of exogenous WT1 isoform WTA on NB4 cell differentiation induced by ATRA. The changes of PML / RARα, p21, c-myc gene expression in cells were detected by RT-PCR. Results After treated with ATRA for 48 hours, NB4 / WTA cells transfected with NB4 / CMV and non-transduced WTA NB4 cells showed obvious differentiation and NB4 / WTA cells only partially differentiated. The relative fluorescence intensity of CD11b in NB4 / WTA cells (4.63 ± 1.63) was lower than that in control group (8.15 ± 1.84) by flow cytometry. The relative fluorescence intensity of CD11b (31.42 ± 5.65) was significantly higher than that in control group (71.95 ± 9.36) The difference is even more obvious. NB4 / WTA cells NBT reduction ability decreased compared with the control group, ATRA treatment NBT reduction between the two groups of cells more obvious differences in ability to reduce. RT-PCR indicated that the expression of PML / RARα, p21, c-myc in NB4 / WTA cells was higher than that in control group. Conclusions Increasing the expression of WTA, an exogenous WT1 isoform, and changing the proportion of WT1 isoform expression from + 17AA / + KTS predominance to -17AA / -KTS predominance can partially inhibit ATRA-induced NB4 cell differentiation Which may be related to the up-regulation of PML / RARα, p21, c-myc gene.