摘要
目的:探讨WT1基因异构体比例的改变对急性早幼粒细胞性白血病细胞株NB4基因表达谱的影响。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+转染入白血病细胞株NB4,建立WT1基因异构体表达比例改变的单克隆细胞株NB4/WTA。运用cDNA微阵列技术检测WT1基因异构体表达比例的转变对NB4细胞基因表达谱的影响。并用RTPCR方法验证cyclinA1及CDK7基因表达的改变。结果:转染WT1基因异构体WTA后,外源基因在mRNA及蛋白水平上稳定表达,NB4细胞中共89个细胞周期相关基因、细胞凋亡相关基因、癌基因以及细胞信号和传递蛋白等基因出现2倍以上表达水平的改变。RTPCR证实与细胞周期相关的cyclinA1基因表达上调,CDK7基因表达下调。结论:WT1基因异构体WTA表达比例增加能引起NB4细胞基因表达谱的改变,这些基因的改变可能对白血病细胞增殖、分化和凋亡等生物学行为产生影响。
Objective： To explore the potential effect of change in proportions of WT1 gene isoforms on gene expression profiles in acute promyelocytic leukemia cell line NB4. Methods：The eukaryotic expression recombinant vector （pCB6 ＋/WTA） containing full-length of human WT1 isoform （WTA：-17AA/-KTS） cDNA and the pCB6 ＋ vector alone were transfected into the leukemia cell line NB4 by electroperforation. The positive cell clones （NB4/WTA） were selected with G418. The WTA mRNA and protein in transfected cells were detected by reversed transcriptase polymerase chain reaction （RT-PCR） and western blotting, respectively, cDNA microarray was used to explore the effect of transfection of exogenous WT1 gene isoforms on gene expression profiles in leukemia NB4 cells. Expression of cyclinA1 and CDK7 genes were determined by semi-quantitative RT-PCR. Results：Exogenous WTA gene isoforms had stable expressions at mRNA and protein levels in NB4 cells after transfection. The gene expression profiles were changed by transfection of exogenous WTA gene isoforms into NB4 cells. Among 4,096 gene clones on the microarray, 89 （2. 17%） genes underwent more than 2.0-fold changes and most of them （nearly 88. 7%） were down-regulated. These genes are cell cycle-related genes, apoptosis-related genes, oncogenes, signal transduction-related genes, transport protein genes, etc. RT-PCR showed that cyclin A1 gene expression was up-regulated and CDK7 gene expression was down-regulated. Conclusion： The introduction and expression of exogenous WTA gene isoforms can alter the gene expression profiles in NB4 cells. Alteration in gene expression profiles had influence on biological behaviors such as growth, differentiation,and apoptosis of leukemia NB4/WTA cells.
出处
《肿瘤》
CAS
CSCD
北大核心
2006年第6期512-518,共7页
Tumor
基金
国家自然科学基金资助项目(编号:39770306)
江苏省卫生厅科研基金资助项目(编号:H9703)
