大萼香茶菜甲素通过线粒体途径诱导HL-60白血病细胞凋亡  

Macrocalyxin A induces apoptosis by mitochondrial signaling pathway in HL-60 leukemia cells

作　　者：吴建国[1] 周永列[1] 夏大静[2] 王珍妮[1] 夏骏[1] 邱莲女[1] 吴茅[1] 林慧君[1] 费鲜明[1] 

机构地区：[1]浙江省人民医院中心实验室 [2]浙江大学免疫学研究所

出　　处：《中国临床药理学与治疗学》2009年第7期741-747,共7页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：浙江省医学重点学科建设基金(07-010);浙江省自然科学基金(Y2080020)

摘　　要：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。AIM： To study the effects of macrocalyxin A （MA） on proliferation inhibition and apoptosis in HL-60 human leukemia cell line and explore its mechanisms. METHODS： Different concentrations of MA and different times of cultivation were used to treat HL-60 cell. The proliferation inhibition was analyzed by MTF assay. The cell apoptosis was analyzed by cell morphology, DNA agarose gel electrophoresis, DNA content and cell cycle analyzation, Annexin-V/PI and Hoechst 33258 fluorescence staining. The expressions of Bcl-2, Bax, Fas P53 and mitochondrial membrane protein were analyzed by flow cytometry, while the mitochondrial transmembrancepotential （Aatrm） was labeled by dihydrorhodamin 123. RT-PCR method was used to study the Bcl-2, Bax, P53 and caspase-3 mRNA levels. RESULTS： MA could inhibit HL-60 cell proliferation viability within a certain range of treating time and dose, with a 24 h IC50 of 8.76 μg/mL, 48 h of 7.17 μg/mL and 72 h of 7.14μg/mL. A majority of HL-60 cells were arrested in G0/G1 phase. The HL-60 cells apoptosis was confirmed by type cell morphology, DNA fragment, sub-G1 phase and Annexin-V/PI labeling method with a time and dose related manner. The expression of Bax was increased, and Bcl-2, P53 and fas were unchanged by the treatment of MA. MA could increase the expression of mitochondrial membrane protein and caspase-3 in a dose-dependent manner while the △ψm was reduced. CONCLUSION： MA can inhibit the proliferation and induce the apoptosis of HL-60 cells. The mechanisms associate with its up-regulation of Bax and the ratio of Bax/Bcl-2, decreasing the mitochondrial membrane potential, opening the mitochondrial membrane pore and activating caspase-3.

关 键 词：大萼香茶菜甲素 白血病 细胞凋亡 线粒体 

分 类 号：R965.2[医药卫生—药理学]