摘要
目的:在明确千金子甾醇诱导HL-60白血病细胞发生凋亡的基础上,从Fas/FasL信号通路的角度探讨其诱导凋亡的分子机制。方法 HL-60细胞培养体系中分别以千金子甾醇终浓度2.5、10、40μg/ml作用24 h后CCK-8法检测千金子甾醇对HL-60细胞增殖的抑制作用,光学及荧光显微镜下观察细胞形态学变化,Annexin Ⅴ/PI 流式细胞术检测细胞凋亡,反转录-聚合酶链反应法检测Fas/FasL、caspase-8和caspase-3 mRNA转录水平,比色法检测caspase-8和caspase-3的相对活性。结果千金子甾醇可明显抑制HL-60细胞的增殖,增殖抑制率分别为(34.9±3.7)%、(54.6±5.2)%、(61.3±4.3)%,细胞呈典型凋亡形态学改变,细胞早期凋亡率分别为(23.4±3.1)%、(35.7±4.3)%、(53.2±3.9)%。Fas、FasL、caspase-8和caspase-3 mRNA转录水平明显上调(P<0.01),caspase-8和caspase-3活性显著增高(P<0.01)。结论千金子甾醇可以诱导HL-60细胞发生剂量依赖性细胞凋亡,其机制与上调Fas/FasL凋亡信号通路有关。
Objective To investigate the effect of euphorbiasteroid on inducing the apoptosis of HL-60 cells and demonstrate whether the Fas/FasL signaling pathway is involved in the induction of apoptosis. Methods HL-60 cells were treated with dose of 2.5,10,40 μg/ml of euphorbiasteroid in vitro for 24 h respectively.After that,cell counting Kit-8 was used to detect cell proliferation.The morphology of HL-60 cells were observed under light and fluorescent microscopy.The early cell apoptosis was detected by using flow cytometry with Annexin Ⅴ-FITC /PI double staining.The expressions of Fas,FasL,caspase-8 and caspase-3 mRNA were analyzed by the method of RT-PCR.The activities of caspase-8 and caspase-3 were examined by chromatometry.Results Compared with 1640 control group,HL-60 cell proliferation was inhibited significant-ly by euphorbiasteroid.The inhibition rates were (34.9 ±3.7)%,(54.6 ±5.2)% and (61.3 ±4.3)%respectively.Moreover,HL-60 cells exhibited typical morphological features.Early cell apoptosis rates of HL-60 cells were (23.4 ±3.1)%,(35.7 ±4.3)% and (53.2 ±3.9)% respectively.Furthermore,the expressions of Fas,FasL,caspase-3 and caspase-8 mRNA were up-regulated significantly after euphorbiasteroid administration in a dose-dependent manner (P<0.01 ).After treated with euphorbiasteroid,the activities of caspase-8 and caspase-3 were significantly enhanced (P<0.01 ).Conclusion The up-regulation effect of euphorbiasteroid on Fas/FasL signaling pathway might contribute to the apoptosis of HL-60 cells.
出处
《国际肿瘤学杂志》
CAS
2014年第9期679-684,共6页
Journal of International Oncology
基金
基金项目:国家“十二五”科技支撑计划项目(2013BA107802)
国家自然科学基金(30771103)
山东省中药现代化重大科技计划项目(2008GG2NS02023)
山东省科技攻关政策扶持项目(2011GGC03083)
山东省科技发展计划项目(ZR2011HL045)
山东省中医药科技发展计划项目(2011-234)
山东中医药大学“名科工程”青年骨干培养计划(ZYDXY1328)