摘要/Abstract
摘要： 目的 ·探讨 HIF-1α-PDK1信号系统调控急性单核细胞白血病细胞糖代谢以及耐药的机制。方法 ·定量聚合酶链式反应（quantitative polymerase chain reaction，qPCR）检测 U937细胞、 U937/DNR细胞和原代急性单核细胞白血病细胞中丙酮酸脱氢酶激酶 1（pyruvate dehydrogenase kinase 1，PDK1）的 mRNA水平。采用基因沉默构建 siRNA HIF-1α质粒，分别转染作用于 U937和 U937/DNR细胞 24 h；噻唑蓝（ MTT）比色法检测细胞增殖抑制， qPCR检测 PDK1 mRNA表达量，蛋白印迹法（ Western blotting）检测 PDK1和多药耐药基因 1（multi-drug resistance gene 1，MDR1）蛋白的表达，流式细胞术检测染料 JC-1水平以评估线粒体膜电位变化，血气分析仪测定培养液中乳酸含量。用二氯乙酸盐（ dichloroacetate，DCA）和柔红霉素（ daunorubicin，DNR）作用于 U937/DNR和原代急性单核细胞白血病细胞 24 h；MTT比色法检测细胞增殖抑制， Western blotting检测 PDK1和 MDR1蛋白的表达。结果 · PDK1 mRNA在原代急性单核细胞白血病细胞、 U937细胞和 U937/DNR细胞中均高表达。沉默低氧诱导因子 -1α（hypoxiainducible factor-1α，HIF-1α）可显著抑制 U937和 U937/DNR细胞的增殖活力、 PDK1和 MDR1蛋白的表达以及乳酸的生成。 DCA能够逆转 U937/DNR细胞和已复发的原代急性单核细胞白血病细胞对 DNR的耐药。结论 · HIF-1α-PDK1信号系统可调控细胞糖代谢并参与急性单核细胞白血病的耐药。
关键词: 丙酮酸脱氢酶激酶 1, 多药耐药, 低氧诱导因子 -1&, alpha, 急性单核细胞白血病
Abstract:
Objective · To investigate the mechanism of HIF-1α-PDK1 signaling system mediated glucose metabolism and drug resistance in acute monocytic leukemia cells. Methods · The of pyruvate dehydrogenase kinase 1 (PDK1) mRNA in U937, U937/DNR and acute monocytic leukemia cells was detectedquantitative polymerase chain reaction (qPCR). siRNA HIF-1α plasmid was constructed and transferred to U937 and U937/ DNR cells for 24 hgene silencing. Cell proliferation inhibition was examinedMTT assay. The level of PDK1 mRNA was detectedqPCR, and the of PDK1 and multi-drug resistance gene 1 (MDR1) proteins was detectedWestern blotting. Cell membrane potential was measuredflow cytometry using JC-1. Lactic acid level in the culture fluid was determinedblood gas analyzer. Dichloroacetate (DCA) and daunorubicin (DNR) were added to treat U937/DNR and acute monocytic leukemia cells for 24 h, MTT was used to calculate cell proliferation inhibition and Western blotting was used to estimate the of PDK1 and MDR1 proteins. Results · PDK1 mRNA was highly expressed in U937, U937/DNR and acute monocytic leukemia cells. Silencing hypoxia-inducible factor-1α (HIF-1α) significantly inhibited the proliferation activity, PDK1 and MDR1 and lactic acid production in U937 and U937/DNR cells. DCA could reverse the resistance to DNR in U937/DNR and relapsed acute monocytic leukemia cells. Conclusion · HIF-1α-PDK1 signaling system may regulate glucose metabolism and participate in the drug resistance of acute monocytic leukemia.
Key words: pyruvate dehydrogenase kinase 1 (PDK1), multidrug resistance, hypoxia-inducible factor-1&, alpha, (HIF-1&, alpha, ), acute monocytic leukemia
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