铜对L-02细胞线粒体的损伤作用

Injury Effects of Copper on Mitochondria of Human Liver Cells L-02

[目的]观察铜对人肝细胞L-02线粒体的损伤作用。[方法]将硫酸铜用D-Hank’s液和培养液稀释成终浓度分别为50、100、150、200μmol/L的硫酸铜与L-02细胞共培养18h,进行量效关系研究;以150μmol/L浓度与L-02细胞共培养0、6、12、18、24h进行时效关系研究。正常对照组:加入与处理组等体积的D-Hank’s液。采用四甲基偶氮唑盐微量酶反应比色法(MTT法)检测线粒体酶抑制率和整体功能的变化;透射电镜观察线粒体超微结构的变化;罗丹明123(Rh123)和碘化丙啶(PI)结合流式细胞仪(FCM)检测线粒体膜电位(MMP)的变化和细胞凋亡率;免疫细胞化学法检测细胞色素C的释放。[结果]铜可引起线粒体酶抑制率和线粒体功能的变化;透射电镜下观察到线粒体结构受到不同程度的损伤,并随剂量增大和时间延长损伤加重;FCM结果表明随着铜剂量增大,实验组Rh123平均荧光强度逐渐降低,由对照组93.60±18.12下降至200μmol/L组59.94±13.55,即膜电位呈逐渐降低趋势;而细胞凋亡率逐渐增加,由对照组2.73%增加至200μmol/L组17.07%(P<0.05,P<0.01)。随着铜作用时间的延长,Rh123平均荧光强度逐渐降低,由对照组93.60±18.12降低至24h组55.74±15.45,即膜电位呈逐渐降低趋势;而细胞凋亡率逐渐增加,由对照组2.73%降低至24h组20.45%(P<0.05,P<0.01)。免疫细胞化学实验显示,细胞色素C从线粒体释放入胞质,释放量呈现一定的剂量依赖性和时间依赖性。[结论]铜可诱导L-02细胞线粒体损伤,引起膜电位降低,细胞色素C释放入胞质,线粒体结构和整体功能遭破坏,导致细胞损伤甚至凋亡。

[Objective] To investigate the injury of mitochondria of human liver cells L-02 caused by copper.[Methods] The function of mitochondrion was determined by MTT assay. Mitochondrial ultrastructure was observed under transmission electron microscope. Rhodamine123（Rh123）and propidium iodide（PI）combining with flow cytometer were used to detect the changes of mitochondrial membrane potential（MMP）and cell apoptosis. Cytochrome C（CtyC）released from mitochondrion to endochylema was observed by immunocytochemistry.[Results] Copper inhibited the function of mitochondrion in a concentration and time dependent manner. Different extent injuries of the mitochondrial ultrastructure were observed under transmission electron microscope. The results of flow cytometer detection indicated fluorescence intensity of Rh123 decreased from 93.60±18.12（control group）to 59.94±13.55（200 μmol/L group）,i.e. mitochondrial membrane potentia（lMMP）decreased in a dose dependent manner （P 0.05,P 0.01）. The rate of cell apoptosis increased from 2.73%（control group）to 17.07%（200 μmol/L group）（P 0.05,P 0.01）. With time extension,Rh123 fluorescence intensity degraded from 93.60±18.12（control group）to 55.74±15.45（24 h group）,i.e. mitochondrial membrane potential（MMP）degraded in a time dependent manner（P 0.05,P 0.01）. The rate of cell apoptosis increased from 2.73%（control group）to 20.45%（24 h group）（P 0.05,P 0.01）. Immunocytochemistry results showed Cty C releasing from mitochondrion to endochylema,and the quantity increased in a concentration and time dependent manner.[Conclusion] Copper could induce damage to mitochondria of human liver cell L-02 and make the MMP reduction and Cty C releasing from mitochondria to endochylema. The structure and function of mitochondria were destroyed. So cells were damaged even caused apoptosis.