蟾毒灵可抑制人红白血病细胞增殖并下调肾母细胞瘤1基因表达

已有研究证实蟾毒灵具有抑制肿瘤细胞增殖及诱导细胞凋亡的作用,在白血病治疗中疗效显著,然而其机制尚未阐明。本研究试图探讨蟾毒灵对人红系白血病(HEL)细胞增殖,肾母细胞瘤基因1 (Wilms'tumor 1 gene, WT1)甲基化的影响及其可能的作用机制。本研究采用不同浓度的蟾毒灵处理HEL细胞,观察细胞形态、增殖情况和细胞周期,采用RT-PCR、Western blotting和免疫细胞化学法检测WT1的mRNA和蛋白表达水平,并用甲基化特异性分析WT1的DNA甲基化和DNA甲基转移酶3a (DNMT3a)的蛋白表达水平。研究结果表明,蟾毒灵对HEL细胞的增殖抑制作用呈剂量依赖性,抑制率为23.13%～84.62%。在蟾毒灵处理的HEL细胞中观察到典型的凋亡形态特征;细胞周期增殖指数由75.45降至49.67;WT1 mRNA及其蛋白表达水平随着蟾毒灵剂量的增加而逐渐降低,同时WT1基因的甲基化状态由未甲基化状态变为部分或完全甲基化状态。而蟾毒灵处理后DNMT3a蛋白的表达水平逐渐增加,呈剂量依赖性。我们的研究初步说明蟾毒灵不仅能显著抑制HEL细胞增殖,阻滞G0/G1期细胞周期,还能诱导细胞凋亡,下调WT1的表达水平。

Bufalin could Inhibit Proliferation of Human Erythroleukemia Cells and Down-Regulate the Expression of Wilms' Tumor 1 Gene

Previous studies have confirmed that bufalin can inhibit the proliferation of tumor cells and induce apoptosis.It has a significant effect in the treatment of leukemia,but its mechanism has not been elucidated yet.This study attempted to explore the effect of bufalin on human erythroleukemia(HEL)cell proliferation,Wilms'tumor 1 gene(WT1)methylation and its possible mechanism.HEL cells were treated with bufalin at different concentrations and cell morphology,proliferation and cell cycle were observed.The mRNA and protein expression level of WT1 were detected by RT-PCR,Western blotting and immunocy to chemistry,DNA methylation and protein expression level of DNA methyltransferase 3a(DNMT3a)of WT1 were analyzed by methylation specificity.The results showed that the inhibitory effect of bufalin on the proliferation of HEL cells was dose-dependent,with the inhibition rate ranging from 23.13% to 84.62%.Typical apoptotic morphology was observed in HEL cells treated with bufalin.Cell cycle proliferation index decreased from 75.45 to 49.67.The mRNA and protein expression level of WT1 decreased graduallywith the increase of bufalin dose,and the methylation status of WT1 gene changed from unmethylated status to partial or exhaustive methylation.However,the expression level of DNMT3a protein gradually increased after treatment with bufalin,which was dose-dependent.Our study preliminarily showed that bufalin could not only significantly inhibit HEL cell proliferation,block G0/G1 cell cycle,but also induce apoptosis and down-regulate the expression level of WT1.