【摘要】 目的异补骨脂素可抑制多种肿瘤细胞增殖,并诱导细胞凋亡,但是异补骨脂素对白血病细胞是否亦具有同样的作用却不明确。本研究旨在探讨异补骨脂素对人急性髓系白血病HL-60细胞增殖和凋亡的影响及其机制。方法采用MTT法检测不同浓度异补骨脂素(10、20、40、80和160μg/mL)作用于HL-60细胞24和48h后对HL-60细胞的增殖抑制作用;光镜观察细胞形态学变化;Annexin V/PI双染后采用流式细胞术检测细胞凋亡的改变;流式细胞术检测细胞周期的变化;分别采用RT-PCR和蛋白质印迹法检测Bcl-2家族Bcl-2和Bax的mRNA水平和蛋白水平表达变化。结果异补骨脂素对HL-60细胞的增殖具有明显的抑制作用,并呈现时间-剂量依赖性。10、20、40、80和160μg/mL异补骨脂素处理24h后,HL-60细胞的抑制率分别为(5.5±4.5)%、(13.7±3.1)%、(20.4±1.9)%、(35.3±5.5)%和(51.7±2.1)%,F=1 465.33,P<0.001;48h后对HL-60细胞的抑制率分别为(21.7±3.2)%、(34.6±5.6)%、(43.3±1.2)%、(61.4±8.76)%和(84.7±4.4)%,F=146.33,P<0.05,差异均有统计学意义。经异补骨脂素处理的HL-60细胞体积明显变小、且有凋亡小体产生,呈现典型的凋亡形态学改变;40μg/mL异补骨脂素处理HL-60细胞24h后细胞早期凋亡率为(16.6±2.7)%,与对照组的(2.8±1.5)%相比,差异有统计学意义,P<0.05;异补骨脂素不引起细胞周期阻滞,G0/G1期前出现典型亚二倍体凋亡峰。RT-PCR和蛋白质印迹法检测结果表明,异补骨脂素可上调Bax、下调Bcl-2的mRNA水平和蛋白水平表达。结论异补骨脂素可抑制人急性髓系白血病HL-60细胞增殖,并诱导其凋亡,其机制与促进Bax蛋白表达和抑制Bcl-2蛋白表达相关。更多还原

【Abstract】 OBJECTIVE Accumulating evidence has demonstrated that angelicin exhibits potent antitumor properties by suppressing cell growth and inducing apoptosis in a variety of cancer cell lines.But whether angelicin exhibits the same effects in leukemia cells has not been clarified.The present study aims to detect the effect of angelicin on proliferation and apoptosis of HL-60 cells and its mechanism.METHODS Cell proliferation was analyzed using MTT assay after HL-60 cells were treated with different concentrations of angelicin(10,20,40,80 and 160μg/mL)for 24 hand 48h.The morphological changes of HL-60 cells were examined by light microscopy.The cell early apoptosis was tested by Annexin V-FITC/PI double-staining analysis.Cell cycle distribution was detected by flow cytometer.RT-PCR and western blot assay were carried out to examine the mRNA and protein expression of Bax and Bcl-2.RESULTS Angelicin inhibited the proliferation of HL-60 cells in a dose-and time-dependent manner.After 10,20,40,80 and 160μg/mL of angelicin treatment for 24 h,the inhibition rate reached to(5.5±4.5)%,(13.7±3.1)%,(20.4±1.9)%,(35.3±5.5)%,(51.7±2.1)%,respectively(F=1 465.33,P<0.001).While after 48 h,the inhibition rate reached to(21.7±3.2)%,(34.6±5.6)%,(43.3±1.2)%,(61.4±8.76)%,(84.7±4.4)%(F=1 46.33,P<0.05).There were significant differences among them(P<0.05).Part of HL-60 cells became small,even formed apoptotic bodies,exhibiting typical morphological features of apoptosis under light micrograph.When HL-60 cells were treated with 40μg/mL of angelicin for 24 h,the ratio of early apoptotic cells reached(16.6±2.7)% vs(2.8±1.5)%in control(P<0.05).Angelicin could not induce cell cycle arrest,but produce a increase in the sub-diploid(apoptotic)cell fraction.RT-PCR and Western blot results shows that treatment of HL-60 cells with angelicin up-regulated the expression of Bax,whereas down-regulated the expression of Bcl-2in a time-dependent manner.CONCLUSION Our foundings show that angelicin can significantly inhibit the proliferation of HL-60 cells and induce the apoptosis of HL-60 cells,which may occur through up-regulation of Bax and down-regulation of Bcl-2.更多还原