雄黄微生物浸出液通过线粒体途径诱导人急性髓系白血病细胞HL60细胞凋亡  

Apoptosis Mechanism of Realgar Bioleaching Solution on HL-60 Cell Line via Mitochondrial Membrane Potential

作　　者：海洋[1] 李建银[1] 王欣 吴争荣[1] 李洋[1] 余兰[1] 谢亲建 李红玉[1,3] HAI Yang;LI Jian-yin;WANG Xin;WU Zheng-rong;LI Yang;YU Lan;XIE Qin-jian;LI Hong-yu(School of Pharmacy,Lanzhou University,Lanzhou 730000,China;Gansu Corps Hospital of CAPF,Lanzhou 730000,China;Institute of Microbiology,School of Life Sciences,Lanzhou University,Lanzhou 730000,China)

机构地区：[1]兰州大学药学院,甘肃兰州730000 [2]武警甘肃省总队医院,甘肃兰州730000 [3]兰州大学生命科学学院,甘肃兰州730000

出　　处：《药物生物技术》2018年第4期283-287,共5页Pharmaceutical Biotechnology

基　　金：国家自然科学基金项目(No.81403145;No.81560715);"重大新药创制"科技重大专项子项目(No.2015ZX09501-004-003-008)

摘　　要：研究雄黄微生物浸出液(RBS)诱导急性髓系白血病细胞HL-60细胞凋亡的作用机制。应用MTT比色法检测不同时间与不同浓度雄黄微生物浸出液对HL-60细胞的抑制作用。应用Hoechst33258染色、流式细胞术、罗丹明123染色以及Western blot等多种方法,研究雄黄微生物浸出液对肿瘤细胞凋亡的作用机制。结果:雄黄微生物浸出液对HL-60细胞增殖有明显的抑制作用,且呈浓度梯度与时间梯度依赖性,阻滞HL-60细胞到G2/M期,促进细胞凋亡以及降低线粒体膜电位。下调cyclin B1、CDK1、c-myb、c-myc、Bcl-2的表达,上调cleaved-caspase3的表达。降低线粒体膜电位,通过线粒体途径激活caspase3,下调癌蛋白c-myc的表达以及降低抗凋亡蛋白Bcl-2可能是RBS对HL-60细胞G2/M阻滞以及诱导凋亡的潜在机制。Realgar bioleaching solution（ RBS） is made by using microbiological leaching method. Previous researches showed that RBS has a significant anti-proliferation effect on chronic myeloid leukemia cells,but the effect and mechanisms of acute myeloid leukemia cells are still not clear. To study anti-proliferation effect of RBS on acute myeloid leukemia cell lines HL-60 and to explore its apoptosis mechanism,HL-60 cells investigated the anti-proliferation effect by MTT assay by treating with RBS after 24,48 and72 h. By Hoechst 33258 dyeing HL-60 cells after treating with RBS in 24 h to explore the apoptotic body. After this,flow cytometry assay has been used to explore the cell cycle arrest and Annexin V/PI dye to explore apoptosis of HL-60. Then studying on the mitochondrial membrane potential level by rhodamine123 dye. At last,detect the protein expression of cyclin B1,CDK2,c-myb,c-myc and Bcl-2 by western blot. RBS has a significant inhibition ratio for HL-60 cells which is shown as concentration-dependent and timedependent,and IC50 are 3. 12,1. 27 and 0. 13 μg/m L in 24,48 and 72 h respectively. HL-60 was apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies was observed by Hoechst 33258 dye.Apoptosis proportion is also be shown by the FCM results. In addition,RBS can also arrest the cell cycle within G2/M. Beyond this,rodamine123 dye demonstrated that RBS can reduce the mitochondrial membrane potential. According to the western blot,the protein expression of cyclin B1,CDK2,c-myc and Bcl-2 were decreased by treating with RBS for 24 h,while c-myb showed a little downtrend. In addition,the protein expression of cleaved-caspase3 was increased within concentration-dependent. RBS has a capacity of cell cycle arrest in G2/M and apoptosis. Reducing the mitochondrial membrane potential,activating caspase3,and down regulating the expression of protein c-myc and Bcl-2 which may be the potential mechanisms.

关 键 词：雄黄 微生物浸出 HL-60 白血病 线粒体膜电位 细胞凋亡 细胞周期 作用机制 

分 类 号：R965[医药卫生—药理学] R73-36[医药卫生—药学]