羟基磷灰石纳米粒子对人肝癌BEL-7402细胞毒性的评价

Evaluation of cytotoxicity of hydroxyapatite nanoparticles on human hepatocellular carcinoma cell line BEL-7402

目的 从抑制细胞生长增殖和诱导细胞凋亡角度评价羟基磷灰石纳米粒子对人肝癌BEL 740 2细胞的细胞毒性。方法 将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 740 2细胞 ,用MTT比色法观察其细胞毒性 ,荧光显微镜、透射电镜检测细胞凋亡形态改变 ,前者同时记数凋亡指数 ,流式细胞仪定量分析细胞凋亡率。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 740 2细胞的生长 ,作用 48h后的半数有效抑制浓度IC50 值为 2 9.3 0 μg/ml。荧光显微镜和透射电镜观察到细胞皱缩、核质浓缩、核碎裂、细胞起泡以及凋亡小体形成等凋亡特性的形态学改变。流式细胞仪直方图上可见亚二倍体峰。浓度为 5 0 μg/ml、75 μg/ml、10 0 μg/ml、15 0 μg/ml、2 0 0 μg/ml的纳米粒子处理 48h后 ,细胞凋亡率分别为 ( 2 0 .3 5± 2 .2 3 ) %、( 2 5 .3 5± 1.92 ) %、( 2 9.3 4± 4.61) % ,( 4 4.92± 3 .78) %和( 5 3 .64± 3 .49) % ,与对照组 ( 2 .2 3± 0 .14 ) %比较均有显著性差异。 5 0 μg/ml浓度作用 12h、2 4h、3 6h和 48h后的细胞凋亡率分别为 ( 2 .73± 0 .17) %、( 3 .5 2± 0 .3 5 ) %、( 6.3 7± 0 .3 4) %和 ( 2 1.64± 2 .46) % ,与对照组 ( 2 .2 3± 0 .14 )

Objective To evaluate the cytotoxicity of hydroxyapatite (HAP) nanoparticles on human hepatocellular carcinoma cell line BEL 7402 by means of growth inhibition and apoptosis induction in vitro. Methods The hepatocellular carcinoma cell line BEL 7402 was treated with HAP nanoparticles at various concentrations for different duration. Growth suppression was evaluated by MTT method; apoptosis related alterations in morphology were ascertained under cytochemical staining (Hoechst 33258) and transmission electron microscopy (TEM). The apoptotic index was counted under fluorescence microscopy. 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, with IC 50 values of 29.30μg/ml after 48h treatment. Under fluorescent staining and transmission electron microscopy, the cells treated with HAP nanoparticles exhibited characteristics of apoptosis including cell membrane shrinkage, cell membrane blebbing, condensation and fragmentation of nuclear chromatin and formation of apoptotic bodies. Flow cytometry analysis showed hypodiploid peaks on histogram, the apoptotic rates at concentrations of 50μg/ml, 75μg/ml, 100μg/ml, 150μg/ml and 200μg/ml were (20.35±2.23)%,(25.35±1.92)%,(29.34±4.61)%,(44.92± 3.78 )% and (53.64±3.49)%, respectively all of which were significantly higher than that of control group (2.23± 0.14)%, but same as (2.73±0.17)%,(3.52±0.35)%,(6.37±0.34)% and (21.64±2.46)% at 12h, 24h, 36h and 48h. Conclusion HAP nanoparticles not only inhibit the proliferation but also induce apoptosis of human hepatocellular carcinoma cell line BEL 7402 with demonstrated strong cytotoxicity.