摘要
目的:探讨环孢霉素A(CsA)对葡萄糖氧化酶(GOX)诱导的肝细胞线粒体功能损伤和细胞凋亡的影响。方法:胞分别给予不同浓度的GOX和CsA处理,MTT法检测细胞活力,确定所要选用的剂量和时间点。以10μmol/L的CsA预处理2 h,并给予25 U/L GOX处理12 h作为CsA+GOX组,同时设立空白对照组、GOX阳性对照组、CsA阴性对照组。分别采用Annexin V/PI双染色结合流式细胞术检测细胞凋亡率;GENMED活体细胞线粒体膜通道孔荧光试剂盒检测线粒体膜通透性转换;JC-1染色激光共聚焦检测线粒体膜电位变化;ATP检测试剂盒检测细胞内ATP水平;液相氧电极法检测线粒体耗氧量。结果:MTT结果显示GOX能诱导HepG2细胞活力下降,且呈现时间和剂量效应关系(P均<0.05);与空白对照组比较,25 U/L GOX作用12 h可诱导HepG2细胞凋亡,同时伴有线粒体膜通道开放和膜电位下降,细胞内ATP水平和线粒体耗氧量下降(P均<0.05),而10μmol/L的CsA预处理2 h有一定的保护作用,可以有效阻断线粒体结构功能的损伤,降低凋亡率,CsA+GOX组与GOX阳性对照组比较差异均具有统计学意义(P均<0.05)。结论:CsA对氧化应激诱导的肝细胞线粒体功能损伤和凋亡具有保护作用。
OBJECTIVE：To explore the protective effects of cyclosporine A on glucose oxidase-induced liver cell apoptosis and mitochondria dysfunction. METHODS：Various concentrations of GOX and CsA were applied to HepG2 cells at different time points, and measured the cell viability using MTT assay, and the appropriate doses and time points selected. Pretreatment of HepG2 cells with 10μmol/L CsA for 2 h,then 25 U/L GOX treatment for 12 h were used as experimental group,and blank control （RPMI-1640 for 14 h）,GOX group as positive control （pretreatment with RPMI-1640 for 2 h,treatment with 25 U/L GOX for 12 h）,CsA group as negative control（pretreatment with 10μmol/L CsA for 2 h,cultured with RPMI-1640 for 12 h）. AnnexinV-FITC apoptosis detection kit was applied to assess apoptosis by flow cytometry;mitochondrial membrane permeability transition was evaluated with the GENMED MPTP fluorescent kits. Mitochondrial membrane potential was observed by means of JC-1 staining and the fluorescence intensity was measured by LSCM. The level of ATP in HepG2 cells was measured by using ATP Assay Kit;and the oxygen consumption was calculated using liquid-phase oxygen measurement system. RESULTS：MTT assay indicated that GOX decreased cell viability in dose and time dependent manners,and 10μmol/L CsA treatment for 2 h had some protective effect. 25 U/L GOX treatment for 12 h resulted in HepG2 cells apoptosis, associated with MPTP opening, the collapse of mitochondrial membrane potential,and the reductions of the ATP level and oxygen consumption. Pretreatment with CsA could prevent structural and functional injuries of the mitochondrial, decreasing apoptosis rate. CONCLUSION：CsA could protect HepG2 cells against mitochondrial dysfunction and apoptosis.
出处
《癌变．畸变．突变》
CAS
CSCD
2014年第1期10-15,共6页
Carcinogenesis,Teratogenesis & Mutagenesis
关键词
葡萄糖氧化酶
环孢霉素A
急性肝损伤
线粒体膜电位
能量供应
glucose oxidase
cyclosporine A
acute liver injury
mitochondrial membrane potential
energy supply
