金纳米粒子对CHO-K1细胞毒性的谷胱甘肽作用机制

目的探讨金纳米粒子(AuNP)对卵巢细胞CHO-K1的毒性作用及谷胱甘肽(GSH)的对抗作用。方法 AuNP 10～100μmol.L-1作用卵巢细胞CHO-K1 72 h,MTT比色法检测细胞存活。AuNP10μmo.lL-1,丁硫氨酸-亚砜亚胺(BSO)20μmo.lL-1及GSH 1 mmo.l L-1单独或联合作用细胞72 h,MTT比色法检测细胞增殖,倒置相差显微镜观察细胞形态,AnnexinⅤ-FITC和PI染色流式细胞仪检测细胞凋亡;AuNP 10μmo.l L-1,BSO 20μmo.l L-1及GSH 1 mmo.l L-1单独或联合作用细胞48 h,共聚焦显微镜检测细胞骨架微丝,JC-1染色流式细胞仪检测线粒体膜电位。结果 AuNP 10～100μmo.lL-1对正常的CHO-K1细胞存活无明显影响。与正常对照组相比,AuNP 10μmol.L-1和BSO 20μmol.L-1联合作用,可明显抑制CHO-K1细胞存活,抑制率为(80±2)%(P<0.01),胞体皱缩、变圆,细胞骨架微丝破坏;凋亡率为(66±6)%(P<0.01);细胞线粒体膜电位显著增加(P<0.01);加入外源性的GSH可逆转AuNP对因细胞GSH水平受抑而产生的细胞毒性。结论 AuNP对CHO-K1细胞损伤可能与GSH水平降低有关。

Glutathione mechanism of gold nanoparticles cytotoxicity in CHO-K1 cells

OBJECTIVE To investigate the toxicity effect of gold nanopartices (AuNP) on Chinese hamster ovary cells CHO-K1 and evaluate the detoxification effect of glutathione (GSH) on AuNP. METHODS CHO-K1 cells were treated with AuNP 10-100 μmol·L^-1 for 72 h. Cell viability was detected by MTT assay. CHO-K1 cells were treated with AuNP 10 μmol·L^-1 and L-buthionine-sulfoximine (BSO) 20 μmol·L^-1 (with or without GSH 1 mmol·L^-1) for 72 h. Cell viability was detected by MTT assay. The cellular morphology was observed under an inverted phase contrast microscope. The cell apoptosis rate was measured by flow cytometry using AnnexinⅤ-FITC and propidium iodide(PI). CHO-K1 cells were treated with AuNP 10 μmol·L^-1 and BSO 20 μmol·L^-1 (with or without GSH 1 mmol·L^-1) for 48 h. Microfilament of CHO-K1 cells was detected using TRITC-phalloidin and analyzed by confocal laser scanning microscopy. Mitochondrial membrane potential in CHO-K1 cells was measured by flow cytometry using JC-1 staining. RESULTS AuNP 10-100 μmol·L^-1 did not cause evident inhibition of the proliferation of CHO-K1 cells. Compared with normal control group, cell viability was significantly inhibited when the cells were treated with BSO 20 μmol·L^-1+AuNP 10 μmol·L^-1. The inhibitory rate was (80±2)% (P<0.01). The cell morphology changed and microfilament was disrupted. The apoptotic rate increased to (66±6)% (P<0.01). The mitochondrial membrane potential was significantly increased. The cytotoxicity of AuNP could be reversed after outside source GSH was added into the system. CONCLUSION The gold nanopartices cytotoxicity in CHO-K1 cells may be related to the discrease of GSH.