纳米银在HepG2和L02细胞中的生物分布及凋亡作用研究

Bio-distribution and effects of apoptosis induced by silver nanoparticles in HepG2 cells and L02 cells

目的探讨纳米银在人肝癌（Hep G2）细胞株及人正常肝（L02）细胞株内的生物分布及引起凋亡作用差异的可能机制。方法将HepG2细胞、L02细胞分别暴露于终浓度为0（对照）、20、40、80、160μg/ml含纳米银的细胞培养液中24、48 h。采用透射电镜（TEM）观察细胞超微结构及颗粒分布;采用流式细胞术检测细胞线粒体膜电位下降细胞比例和活性氧（ROS）水平的变化。结果染毒24 h时,HepG2和L02细胞的细胞膜受损,在膜周围可见黑色颗粒;染毒48 h时,在HepG2细胞的线粒体及L02细胞的溶酶体和内吞小泡中发现黑色颗粒。与对照组相比,20-160μg/ml纳米银暴露Hep G2细胞24、48 h以及40、80、160μg/ml纳米银暴露L02细胞24 h及80、160μg/ml纳米银暴露L02细胞48 h时的线粒体膜电位下降细胞比例均较高,差异有统计学意义（P〈0.05,P〈0.01）;且随着纳米银暴露浓度的升高暴露时间的延长,HepG2细胞和L02细胞的线粒体膜电位下降细胞比例均呈上升趋势。与对照组相比,20-160μg/ml纳米银暴露HepG2细胞24、48 h的ROS水平上升明显,差异有统计学意义（P〈0.01）;且随着纳米银暴露浓度的升高暴露时间的延长,HepG2细胞ROS水平呈上升趋势;各剂量纳米银暴露L02细胞中ROS水平无明显变化。结论纳米银在HepG2和L02细胞中的生物分布不同对这两种细胞的凋亡造成了不同的影响。

Objective To investigate the bio-distribution of silver nanoparticles in human hepatoma cell line（HepG2） cells and human normal liver cell line（L02） cells in vitro and the possible mechanism of apoptosis induced by silver nanoparticles.Methods The HepG2 cells and the L02 cells were used. The two cell lines were treated with silver nanoparticles at the level of 20-160 μg/ml for 24 h and 48 h in vitro. The cell morphology and particle distribution in cells were observed by transmission electron microscopy（TEM）. The changes of mitochondrial membrane potential and the production of reactive oxygen species（ROS） were determined by flow cytometry assay. Results After 24 h of treatment,impaired cell membranes and black particles around the membrane were found in both HepG2 cells and L02 cells. After 48 h of treatment,black particles could be detected in the mitochondria of HepG2 cells,while in the lysosome and endocytic vesicle of L02 cells. Compared with the control group,HepgG2 cells treated with 20-160 μg/ml silver nanoparticles for 24 h and 48 h groups,L02 cells treated with40,80,160 μg/ml silver nanoparticles for 24 h groups and 80,160 μg/ml silver nanoparticles for 48 h groups showed a higher ratio of the cells whose mitochondrial membrane potential reducing（P〈0.05,P〈0.01）. With the increased exposure concentration and the extended exposure time,the ratio showed an upward trend. Compared with the control group,HepgG2 cells treated with20-160 μg/ml silver nanoparticles for 24 h and 48 h groups showed a higher ROS level（P〈0.05,P〈0.01）. With the increased exposure concentration and the extended exposure time,the ROS level showed an upward trend. Conclusion The different bio-distribution of silver nanoparticles may cause the different effects of apoptosis in HepG2 cells and L02 cells.