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张雪莲,周尊海,张利强,等.As₂O₃诱导NB4、HL-60细胞凋亡的机制研究[J].同济大学学报（医学版）,2016,37(3):8-15, 27. [点击复制]
ZHANG Xue-lian,ZHOU Zun-hai,ZHANG Li-qiang,et al.Mechanisms of arsenic trioxide-induced apoptosis in NB4 and HL60 cells[J].同济大学学报（医学版）,2016,37(3):8-15, 27. [点击复制]

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As₂O₃诱导NB4、HL-60细胞凋亡的机制研究
张雪莲,周尊海,张利强,王博,贾玉芳,梁爱斌
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(同济大学附属杨浦医院内分泌科,上海 200090;上海赛金生物医药有限公司,上海 201203;济宁市第一人民医院妇产科,山东济宁 272011;同济大学附属同济医院血液科,上海 200065)

摘要:

目的探讨三氧化二砷(As₂O₃)在体外抑制急性早幼粒白血病(acute promyelocyticleukemia, APL)细胞NB4、HL-60细胞增殖,诱导细胞凋亡的分子机制。方法通过WST-8法检测上述两个细胞株的增殖抑制曲线;用AnnexinV/PI流式细胞术检测细胞的凋亡;用Real time PCR检测凋亡相关基因Caspase-3、Caspase-8、Caspase-9、Bcl-2、Bax、p53、C-myc、Survivin在As₂O₃处理前后的表达变化,同时用Westernblot检测凋亡相关蛋白Caspase-3、Caspase-8、Caspase-9、Bcl-2、Bax、P53在As₂O₃处理前后的表达变化。结果 As₂O₃能够显著抑制两种细胞增殖, Westernblot检测及Realtime PCR结果显示,As₂O₃处理引起了两种细胞中Bax、Caspase-3、Caspase-8 、Caspase-9基因表达水平增加,且蛋白表达水平上都出现了活性形式的Caspases。NB4细胞中Survivin、C-myc、Bcl-2的基因表达水平都显著降低,突变型p53蛋白在细胞内的量同样显著下降；HL-60细胞的C-myc表达水平显著降低,但Survivin、Bcl-2表达水平无明显变化。结论 As₂O₃能在药物临床浓度范围内有效抑制急性早幼粒白血病细胞HL-60、NB4的细胞增殖, 但方式不同；1.5μmol/L浓度的As₂O₃对NB4细胞生长的抑制体现在诱导细胞分化并诱导细胞凋亡,对HL-60细胞生长的抑制只体现在诱导细胞分化。HL-60细胞中高表达的Survivin、Bcl-2抑制了细胞的凋亡。NB4、HL-60细胞株中P53基因的细胞遗传学变异的不同可能是As₂O₃对这两种细胞增殖抑制机制差异的主要原因之一。

关键词: As₂O₃ 细胞凋亡 NB4 HL-60 survivin p53

DOI：10.16118/j.1008-0392.2016.03.002

投稿时间：2015-09-12

基金项目:国家自然科学基金(81270615)；上海市卫生局科研基金(20134470)

Mechanisms of arsenic trioxide-induced apoptosis in NB4 and HL60 cells

ZHANG Xue-lian,ZHOU Zun-hai,ZHANG Li-qiang,WANG Bo,JIA Yu-fang,LIANG Ai-bin

(Dept.of Endocrinology, Yangpu Hospital, Tongji University, Shanghai 200090, China;Shanghai Cellgene bio Pharmaceutical Co.Ltd., Shanghai 201203, China;Dept.of Gynecology, Jining First People's Hospital, Jining 272011, Shandong Province, China;Dept.of Hematology, Tongji Hospital, Tongji University, Shanghai 200065, China)

Abstract:

Objective To investigate the mechanism of arsenic trioxide (As₂O₃) -induced apoptosis in acute promyelocytic leukemia(APL)NB4 and HL60 cells.Methods NB4 and HL60 cells were treated with As₂O₃ at different concentrations. Cell proliferation was determined by WST-8 assay. The apoptosis rate was detected by flow cytometry with annexinV-FITC/PI double staining.The mRNA expression of apoptosis-related genes caspase-3, Caspase-8, Caspase-9, Bcl-2,Bax, the proto-oncogene C-myc, survivin, p53 and granulocytes differentiation antigen CD11b was detected by RT-PCR. The expression of apoptosis-related proteins caspase-3, Caspase-8, Caspase-9, Bcl-2,Bax was determined by Western blotting. Results Arsenic trioxide proliferation of NB4 and HL60 cells with similar sensitivity. As₂O₃ induced apoptosis of NB4 cells, but induced HL-60 differentiation to mature stage. Western blot detection and RT-PCR results showed that As₂O₃ treatment caused up-regulation of Bax, caspase-3, Caspase-8, Caspase-9, CD11b mRNA levels, and protein levels of caspase-3, Caspase-8, Caspase-9 in both cell lines. The expression of survivin, C-myc, Bcl-2 and mutated p53 in NB4 cells treated with As₂O₃ significantly decreased. The expression of C-myc in HL60 cells treated with As₂O₃ significantly decreased, but survivin and Bcl-2 expression levels were not changed. Conclusion Arsenic trioxide can effectively inhibit proliferation of NB4 and HL60 cells, and induce apoptosis of NB4 cells, while induce HL-60 differentiation to mature stage. These effects may be associated with the altered expression of survivin, Bcl-2 and mutated p53 genes.

Key words: arsenic trioxide cell apoptosis NB4 HL-60 survivin p53