羟基磷灰石纳米粒子诱导人肝癌细胞凋亡模型的构建

Establishment of apoptotic model induced by hydroxyapatite nanoparticles in human hepatoma cell line BEL-7402

目的:建立羟基磷灰石纳米粒子体外诱导人肝癌细胞凋亡的模型,为进一步研究纳米粒子诱导肝癌细胞凋亡的分子机制奠定基础.方法:用羟基磷灰石纳米粒子以不同终浓度、不同时间作用于人肝癌BEL—7402细胞,用细胞毒性实验(MTT比色法)观察其细胞毒性,倒置相差显微镜、荧光显微镜、透射电镜、DNA琼脂糖凝胶电泳等方法观察凋亡在形态学和生化方面的变化.流式细胞仪分析以进一步了解凋亡发生的时间和程度.结果:羟基磷灰石纳米粒子以剂量依赖和时间依赖的方式抑制 BEL-7402细胞的生长.50-200 mg/L的纳米粒子处理 48 h后,形态学上,肝癌细胞表现为细胞皱缩、核质浓缩、核碎裂、细胞起泡以及凋亡小体形成等凋亡特征.琼脂糖凝胶电泳观察到DNA“梯带”.流式细胞仪定量分析,0,50、75、100、150、200 mg/L浓度下凋亡率分别为2.2％,20.3％,25.3％,29.8％,45.1％和53.1％.50 mg/L作用 12 h后肝癌细胞出现凋亡,48 h达高峰,12,24,36,48 h 细胞凋亡率分别为 2.7 ％,3.5％,6.3％和 21.4％.结论:羟基磷灰石纳米粒子既能抑制人肝癌BEL-7402细胞增生,又能诱导其凋亡,该凋亡模型的成功建立将有助于进一步探讨纳米粒子诱导肝癌细胞凋亡的分子机制.

AIM: To establish an apoptotic model induced by hydroxya- patite(HAP) nanoparticles in human hepatoma cell line in vitro in order to explore the mechanism of nanoparticles- induced apoptosis. METHODS: The selected human hepatoma cell line BEL-7402 was treated with HAP nanoparticles at different concentra- tions and for various durations of time. Growth suppression was evaluated by MTT method. Apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33 258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Flow cytometry (FCM) was used to investigate the apoptotic rate. RESULTS: HAP nanoparticles inhibited the growth of hepatoma cells in a dose and time-dependent manner. After BEL-7402 cells were treated with 50-200 mg/L HAP nanoparticles for 48 h, apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage were observed using fluorescent staining and TEM. DNA ladder could be demonstrated on DNA electrophoresis. By FCM analysis, the apoptotic rates at concentrations of 0 mg/L, 50 mg/L, 75 mg/L, 100 mg/L, 150 mg/L, 200 mg/L were 2.2 %, 20.3 %, 25.3 %, 29.8 %, 45.1 % and 53.1 %, respectively. Cell apoptosis began approximately at 12h after administration of 50 mg/L and peaked at 48 h following treatment. The rates at 12, 24, 36, and 48 h were 2.7 %, 3.5 %, 6.3 %, and 21.4 %, respectively. CONCLUSION: HAP nanoparticles not only inhibit prolif- eration but also induce apoptosis of human hepatoma cell line BEL-7402 in vitro. The successful establishment of this model will help further explore the molecular mechanism of hepatoma cells apoptosis.