探讨流式细胞仪测定不同浓度维生素K3对肝癌细胞凋亡周期的影响  

Determining the influence of vitamin K3 at different concentrations on hepatoma cells apoptosis cycle by flow cytometry

作　　者：李宁[1] 于力方[1] 王珊[1] 廖杰[1] 

机构地区：[1]解放军总医院医学实验测试中心,北京100853

出　　处：《海南医学院学报》2012年第10期1372-1374,1378,共4页Journal of Hainan Medical University

基　　金：中国高校医学期刊临床专项资金项目(112210337)~~

摘　　要：目的:对维生素K3诱导肝癌细胞分子凋亡机制进行初步探讨从而达到指导临床肝癌治疗的作用。方法:对人类肝癌细胞株HCC-9204进行培养并选取对数生长期的肝癌细胞,不同浓度及时间的维生素K3进行诱导,通过荧光染色法在激光共聚焦显微镜下观察凋亡细胞核形态。CKK-8法对维生素K3抑制HCC-9204细胞增值,使用流式细胞仪检测细胞凋亡率、凋亡周期及细胞膜Fas表达情况。结果:经维生素K3进行诱导CCK-8检验肝癌细胞比率明显下降,自身生长抑制率上升且呈现明显的计量依赖性,荧光染色后发现细胞凋亡中期细胞凋亡细胞核呈现波纹状或折缝样,有部分染色质出现浓缩,中晚期凋亡细胞核染色质呈现高度凝聚且边缘化,细胞核裂解呈现碎块状并产生凋亡小体。流式细胞仪检测不同浓度VK3作用的肝癌细胞珠HCC-9204在48h时2μmol/L凋亡率为31.44%,5μmol/L凋亡率为62.81%,10μmol/L凋亡率为76.89%,20μmol/L凋亡率为89.24%,25μmol/L凋亡率为93.58%。结论:维生素K3诱导肝癌细胞凋亡48h时呈现明显浓度计量依赖性,荧光染色法在能动速度态观察差细胞核凋亡变化并可以进行定量、定性分析。Objective. To explore the mechanism of vitamin K3-induced hepatoma ceils apoptosis so as to guide the clinical treatment of liver cancer. Methods. Logarithmic phase of liver cancer cells were selected from cultured human hepatoma cell line HCC-9204, and were treated with different concentrations of vitamin K3 for different length of time. Apoptotic nuclear morphology was observed under confocal laser scanning microscope. CKK-8 method was used to detect inhibition of HCC-9204 cell proliferation by vitamin K3, flow cytometry was applied to detect apoptosis rate and apoptosis cycle and cell membrane ex- pression of Fas. Results：CCK-8 test showed that hepatoma cell ratio was significantly decreased; growth inhibition rate increased showing an significant measurement dependence. After staining, apoptotic nuclei in metaphase cells presented a corrugated or crease-like shape, parts of the chromatin condensed, late ap- optotic nuclear chromatin presented a high degree of cohesion and marginalized, chunky nucleus cracked and produced apoptotic bodies. Flow cytometry showed 48 hours after the interference of VK3 at 2μmol/L leaded to apoptosis rate of 31. 44%, 5μmol/L VK3 induced apoptosis rate of 62. 81%, 10 μmol/L, 76.89% ;20μmol/L, 89.24%; 25μmol/L, 93.58%0. Conclusions：Vitamin K3-induced hepatoma cell apoptosis shows the dependence of apparent concentration measurement, the fluorescence staining can reveal dynamic changes in the nuclei apoptosis and can be used for both quantitative, qualitative analysis in this study.

关 键 词：肝癌 细胞凋亡 维生素K3 流式细胞仪 

分 类 号：Q255[生物学—细胞生物学]