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目的:观察大萼香茶菜甲素(macrocalyxinA,MA)体外诱导HL-60细胞凋亡,并探讨其作用机制。方法:不同浓度的MA与HL-60细胞进行培养,采用MTT比色法观察其对HL-60细胞增殖的抑制作用;细胞形态学、DNA含量、DNA梯度电泳及细胞周期分析、Annexin-V/PI双标记和Ho-echst 33258荧光染色等分析其促细胞凋亡效应;流式细胞术和RT-PCR分别检测Bcl-2、Bax、P53、Fas、线粒体膜蛋白(Apo2.7)、线粒体跨膜电位(ΔΨm)与Bcl-2、Bax、P53、caspase-3 mRNA变化水平,研究其促凋亡机制。结果:MA呈现作用时间和剂量依赖性地抑制HL-60细胞增殖和活力;HL-60细胞经MA作用后,Wright-Giemsa染色和Ho-echst荧光染色后细胞出现典型的凋亡小体,细胞阻滞于G0/G1期,DNA片段化,亚二倍体明显增高,Annexin-V/PI标记升高;MA诱导HL-60细胞凋亡过程中,Bcl-2、Fas、P53表达无明显变化,Bax、线粒体膜蛋白(Apo2.7)、caspase-3表达显著增加,Bax/Bcl-2比值升高,ΔΨm下降。结论:MA能抑制HL-60细胞增殖和细胞活力、诱导细胞凋亡,其机制通过上调Bax基因和Bax/Bcl-2比值,使线粒体膜电位下降、膜通透性增高,最终使caspase-3激活而促进凋亡。
Objective: To observe the apoptosis of HL-60 cells induced by macrocalyxin A (MA) in vitro and its mechanism. Methods: Different concentrations of MA and HL-60 cells were cultured. MTT assay was used to observe the inhibitory effect on the proliferation of HL-60 cells. Cell morphology, DNA content, DNA gradient electrophoresis and cell cycle analysis, Annexin-V / PI double labeling and Ho-echst 33258 fluorescence staining were used to detect the pro-apoptotic effect. The expressions of Bcl-2, Bax, P53, Fas, mitochondrial membrane protein (Apo2.7) Membrane potential (ΔΨm) and Bcl-2, Bax, P53, caspase-3 mRNA levels to study its mechanism of apoptosis. RESULTS: MA showed a time-and dose-dependent inhibition of proliferation and viability of HL-60 cells. After treated with MA, HL-60 cells showed typical apoptotic bodies after Wright-Giemsa staining and Ho-echst staining. In the G0 / G1 phase, DNA fragmentation and sub-diploid were significantly increased, while Annexin-V / PI markers were increased. The expression of Bcl-2, Fas and P53 in MA-induced HL-60 cells showed no significant changes , Bax, mitochondrial membrane protein (Apo2.7), caspase-3 expression was significantly increased, Bax / Bcl-2 ratio increased, ΔΨm decreased. CONCLUSION: MA can inhibit the proliferation and cell viability of HL-60 cells and induce apoptosis. The mechanism may be that by increasing the ratio of Bax gene and Bax / Bcl-2, MA decreases the mitochondrial membrane potential and increases membrane permeability, Activate and promote apoptosis.