大萼香茶菜甲素体外诱导白血病细胞株HL-60的分化和凋亡

Study on the differentiation and apoptosis of HL-60 leukemia cells induced by MA in vitro

目的研究大萼香茶菜甲素(macrocalyxin A,MA)抑制白血病细胞株HL-60增殖,诱导细胞分化和凋亡作用,并对其作用机制进行初步探讨。方法将不同浓度的MA与HL-60细胞在体外培养,台盼蓝染色、MTT比色法观察对HL-60细胞增殖的抑制作用;细胞形态学、DNA含量及细胞周期分析、Annexin-V/PI双标记和Hoechst 33258荧光染色等分析细胞凋亡;通过细胞表面抗原CD11b、CD13、CD14,NBT试验和细胞形态学检测MA对HL-60细胞的分化作用;流式细胞术检测Bcl-2、Bax、P53、Fas和线粒体膜蛋白、线粒体跨膜电位(ΔΨm)的表达变化。结果MA呈时效及量效性地抑制HL-60细胞的增殖和活力,8μg/ml MA作用HL-60细胞24～72 h后,MTT法检测24h、48 h、72 h的细胞增殖抑制率分别为(52.9±0.3)%、(70.0±1.0)%和(75.3±1.2)%,均显著高于对照组(P【0.01);24h、48 h、72 h的IC50分别为8.76、7.17、7.14μg/ml;HL-60细胞经MA作用后,大部分细胞阻滞于G0/1期,出现典型的细胞形态改变,经4～20μg/ml的MA作用后,SubG1由(2.85±1.6)%上升至(51.7±5.9)%,与对照组(1.74±0.5)%相比具有显著性差异(P【0.05),Annexin-V/PI标记升高,Hoechst33258染色后的凋亡细胞的特征性改变等均表明MA能诱导HL-60细胞凋亡;HL-60细胞经4～8μg/ml MA作用24h后,细胞发生部分分化,细胞表面CD11b表达增加,NBT阳性细胞增多。MA诱导HL-60细胞凋亡和分化过程中,Bcl-2表达无变化,但Bax表达显著增加,Bax/Bcl-2的比值升高,Fas、P53表达无变化。随MA作用的浓度增加,线粒体膜电位(ΔΨm)下降、而线粒体膜蛋白表达显著升高。结论MA能抑制HL-60细胞增殖和细胞活力、诱导HL-60细胞向粒系分化、促进细胞凋亡,其机制与上调bax基因和bax/bcl-2比值、提高线粒体膜通透性与下调线粒体膜电位等有关。

Objective To study the effect of macrocalyxin A(MA) on proliferation inhibition,differentiation and apoptosis in HL-60 human leukemia cell line and explore its possible mechanisms.Methods Different concentration of MA and the different time of cultivation were used to treat HL-60 cell.The proliferation inhibition was analyzed by trypan blue staining and MTT assay.Cell apoptosis was analyzed by cell morphology,DNA content and cell cycle analyzation,Annexin-V/PI,Hoechst 33258 fluorescence staining.The cell morphological analysis,NBT tests,and expression of CD11b、 CD13 and CD14 were performed to evaluate differentiation of HL-60 cells.The expressions of bcl-2,bax,Fas,P53 and mitochondrial membrane protein were analyzed by flow cytometry,while the mitochondrial transmembrancepotential(ΔΨm) was labeled by dihydrorhodamin 123.Results MA could inhibit HL-60 cell proliferation viability within a certain range of treating time and doses,with a 24h IC50 of 8.76 μg/ml,48 h of 7.17μg/ml and 72h of 7.14 μg/ml.A majority of HL-60 cells were arrested in G0/G1 phase.The HL-60 cells apoptosis was confirmed by type cell morphology,sub-G1 phase and Annexin-Ⅴ/PI labeling method with a time and dose related manner.The morphology of HL-60 cells cultured in the presence of 4-8 μg/ml MA for 24hs was more mature with higher positive rate of NBT and up-regulated expressions of CD11b than those cultured without MA.The expression of bax was increased,and bcl-2 and p53 were unchanged by the treatment of MA.MA could increase the expression of mitochondrial membrane protein in a dose-dependent manner while the ΔΨm was reduced.Conclusion MA can inhibit proliferation,induce differentiation and apoptosis of HL-60 cells.The mechanism may associate with its up-regulation of bax,unwrap of the mitochondrial permeabilitytransition pore and reduction of ΔΨm.