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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 effects of macrocyxin A (MA) on the proliferation, differentiation and apoptosis of leukemia cell line HL-60 and to explore its mechanism. Methods The inhibitory effects of different concentrations of MA and HL-60 cells on the proliferation of HL-60 cells were observed by trypan blue staining and MTT assay. The morphological changes, DNA content and cell cycle analysis, Annexin-V / PI double labeling and Hoechst 33258 fluorescence staining, the differentiation of HL-60 cells was detected by cell surface antigen CD11b, CD13, CD14, NBT assay and cell morphology; Bcl-2, Bax, P53, Fas and mitochondrial membrane protein, mitochondrial transmembrane potential (ΔΨm) expression changes. Results MA inhibited the proliferation and viability of HL-60 cells in a dose-and time-dependent manner. After treated with 8μg / ml MA for 24-72 hours, the cell proliferation inhibition rates of HL-60 cells were detected by MTT assay at 24, 48 and 72 h (52.9 ± 0.3)%, (70.0 ± 1.0)% and (75.3 ± 1.2)%, respectively, were significantly higher than those in the control group (P <0.01). The IC50 values at 24h, 48h and 72h were 8.76,7.17,7.14 After treated with 4 ~ 20μg / ml MA, most of the cells arrested in G0 / 1 phase showed typical morphological changes of cells. After treated with 4 ~ 20μg / ml MA, the percentage of SubG1 was (2.85 ± 1.6)% (51.7 ± 5.9)%, which was significantly higher than that in control group (1.74 ± 0.5)% (P <0.05), and the Annexin-V / PI marker increased. The characteristic changes of apoptotic cells after Hoechst33258 staining All showed that MA could induce the apoptosis of HL-60 cells. When HL-60 cells were treated with 4 ~ 8μg / ml MA for 24 hours, the cells were partially differentiated, the expression of CD11b on the surface of the cells increased and NBT positive cells increased. Bcl-2 expression did not change in MA-induced apoptosis and differentiation of HL-60 cells, but the expression of Bax was increased, the ratio of Bax / Bcl-2 was increased, while the expression of Fas and P53 was unchanged. With the increase of MA concentration, the mitochondrial membrane potential (ΔΨm) decreased, while the mitochondrial membrane protein expression increased significantly. Conclusion MA can inhibit the proliferation and cell viability of HL-60 cells, induce the differentiation of HL-60 cells into granulocytes, and promote the apoptosis of cells. The mechanism of MA is to upregulate the ratio of bax gene and bax / bcl-2 and improve the mitochondrial membrane permeability and down-regulation Mitochondrial membrane potential and so on.