CAJ | 학술논문

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：HepG2细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AnnexinV/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。
목적：탐토배포매소A(CsA)대포도당양화매(GOX)유도적간세포선립체공능손상화세포조망적영향。방법：HepG2세포분별급여불동농도적GOX화CsA처리，MTT법검측세포활력，학정소요선용적제량화시간점。이10μmol/L적CsA예처리2 h，병급여25 U/L GOX처리12 h작위CsA+GOX조，동시설립공백대조조、GOX양성대조조、CsA음성대조조。분별채용AnnexinV/PI쌍염색결합류식세포술검측세포조망솔；GENMED활체세포선립체막통도공형광시제합검측선립체막통투성전환；JC-1염색격광공취초검측선립체막전위변화；ATP검측시제합검측세포내ATP수평；액상양전겁법검측선립체모양량。결과：MTT결과현시GOX능유도HepG2세포활력하강，차정현시간화제량효응관계(P균<0.05)；여공백대조조비교，25 U/L GOX작용12 h가유도HepG2세포조망，동시반유선립체막통도개방화막전위하강，세포내ATP수평화선립체모양량하강(P균<0.05)，이10μmol/L적CsA예처리2 h유일정적보호작용，가이유효조단선립체결구공능적손상，강저조망솔，CsA+GOX조여GOX양성대조조비교차이균구유통계학의의(P균<0.05)。결론：CsA대양화응격유도적간세포선립체공능손상화조망구유보호작용。
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.