摘要
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 study the potential effects of exogenous WT1 gene isoform on the differentiation of leukemia cell line NB4 and its possible molecular mechanisms. Methods The recombinant eukaryotic expression vector （ pCB6 ＋/WTA） containing full-length human WT1 isoform （ WTA ： - 17AA/- KTS） cDNA and the blank pCB6 ＋ vector were transfected into leukemia cell line NB4 by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Cell morphology, NBT reduction and CD1 lb antigen expression in NB4 cells were assayed to evaluate cell differentiation. Expression of PML/RARα,p21 and c-mye genes was determined by semi-quantitative RT-PCR after transfection. Results Compared with NB4/WTA cells, NB4 and NB4/CMV（NB4 cells transfected with pCB6 ＋ vector） cells had higher morphological differentiation rates and higher CD11b expression levels after exposure to ATRA for 48 hours. The percentage of NBT reduction in NB4/WTA cells was lower than that in control groups. The difference in NBT reduction rate between NB4/WTA and control cells was gradually increased after treated with ATRA for three days. The expression levels of PML/RARα,p21 and c-myc genes in NB4/WTA cells were notably increased. Conclusion Overexpression of exogenous WTA gene could partially inhibit the differentiation of NB4 ceils by up-regulating the expression of PML/RARα, p21 and c-myc genes.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2005年第9期543-547,共5页
Chinese Journal of Hematology
基金
国家自然科学基金资助项目(39770306)
江苏省卫生厅科研基金资助项目(H9703)
