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

中国临床药理学与治疗学 ›› 2009, Vol. 14 ›› Issue (7): 741-747.

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

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

¹浙江省人民医院中心实验室;
²浙江大学免疫学研究所, 杭州310014, 浙江

收稿日期:2009-04-17 修回日期:2009-06-18 出版日期:2009-07-26 发布日期:2020-10-30
通讯作者: 周永列, 男, 主任医师, 研究方向:实验血液学及肿瘤细胞凋亡。E-mail:zyl@zjyxjy.com
作者简介:吴建国, 男, 医学硕士, 副主任检验师, 研究方向:实验血液学。E-mail:labzyl@126.com
基金资助:
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)

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

WU Jian-guo¹, ZHOU Yong-lie¹, XIA Da-jing², XIA Jun¹, QIU Lian-nu¹, WU Mao¹, LIN Huijun¹, FEI Xian-ming¹

¹Center Laboratory, Zhejiang Provincial People's Hospital;
²Research Institute of Immunology, Zhejiang University, Hangzhou 310014, Zhejiang, China

Received:2009-04-17 Revised:2009-06-18 Online:2009-07-26 Published:2020-10-30

摘要/Abstract

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Hoechst33258 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Hoechst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。

关键词: 大萼香茶菜甲素, 白血病, 细胞凋亡, 线粒体

Abstract: 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 MTT 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 (ΔΨm) 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 7.14 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, 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 ΔΨm 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.

Key words: macrocalyxin A, leukemia, apoptosis, mitochondria

中图分类号:

R965.2

吴建国, 周永列, 夏大静, 王珍妮, 夏骏, 邱莲女, 吴茅, 林慧君, 费鲜明. 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[J]. 中国临床药理学与治疗学, 2009, 14(7): 741-747.

WU Jian-guo, ZHOU Yong-lie, XIA Da-jing, XIA Jun, QIU Lian-nu, WU Mao, LIN Huijun, FEI Xian-ming. Macrocalyxin A induces apoptosis by mitochondrial signaling pathway in HL-60 leukemia cells[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2009, 14(7): 741-747.

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