【摘要】 [目的]研究铅对人肾小管上皮细胞(human kidney cells,HK-2)线粒体的损伤作用,并探讨其可能的作用机制。[方法]以不同浓度的乙酸铅处理体外培养的HK-2细胞,罗丹明123(rhodamine 123,Rh123)及10-壬基吖啶橙(10-nonyl acridine orange,NAO)结合荧光化学发光仪分别检测细胞线粒体膜电位及心磷脂水平,细胞免疫荧光法结合流式细胞术检测线粒体细胞色素c(cytochrome c,Cyt c)释放情况,加甘露醇观察铅对心磷脂及Cyt c变化的影响。[结果]乙酸铅能造成HK-2细胞线粒体膜电位剂量及时间依赖性下降,荧光强度从正常对照组的4.56±0.25下降到400μmol/L组的2.90±0.26或从正常对照组的4.44±0.20下降到12 h组的2.34±0.50,与正常对照组比较差异有统计学意义(P<0.05)。乙酸铅能明显造成HK-2细胞线粒体心磷脂NAO荧光强度时间及剂量依赖性下降,荧光强度从正常对照组的2.45±0.18下降到400μmol/L组的0.91±0.18或从正常对照组的2.52±0.01下降到24 h组的1.50±0.05,与正常对照组比较差异有统计学意义(P<0.05)。乙酸铅能诱发线粒体Cyt c释放,剂量及时间依赖性地降低细胞内Cyt c荧光强度。50μmol/L甘露醇能使NAO荧光强度从1.38±0.14恢复至2.30±0.15,将Cyt c荧光强度从9.49±0.31恢复至14.20±0.39,明显抑制铅对心磷脂的氧化及Cyt c的释放。[结论]铅可能早期通过HK-2细胞线粒体膜电位溃散,造成线粒体功能损害,进一步加强线粒体心磷脂的氧化损伤,促使Cyt c释放,造成线粒体的结构损伤。更多还原

【Abstract】 [Objective] To explore the possible mechanism of mitochondria damage in human kidney cells(HK-2 cells) induced by lead acetate in vitro.[Methods] Cultured HK-2 cells were exposed to different level of lead acetate.Mitochondrial membrane potential(MMP) and diphosphatidyl glycerol were detected by rhodamine 123(Rh123) and 10-nonyl acridine orange(NAO).The release of cytochrome c(Cyt c) from mitochondria was detected by flow cytometry combined with immunofluorescence.Changes of diphosphatidyl glycerol and Cyt c were observed with mannitol treatment.[Results] After exposed to lead acetate,MMP was descended in a dose-and time-dependent manner in HK-2 cells,significantly down from 4.56±0.25 in normal control group to 2.90±0.26 in 400 μmol/L exposure group and from 4.44±0.20 in normal control group to 2.34±0.50 in 12 h exposure group(P < 0.05).The NAO fluorescence intensity(propidium iodide) of diphosphatidyl glycerol was declined in a dose-and time-dependent manner,significantly down from 2.45±0.18 in normal control group to 0.91±0.18 in 400 μmol/L exposure group and from 2.52±0.01 in normal control to 1.50±0.05 in 24 h exposure group(P < 0.05).Lead acetate induced Cyt c releasing from mitochondria and reduced the fluorescence intensity of Cyt c in a dose-and time-dependent manner.The treatment with 50 μmol/L mannitol recovered the fluorescence intensity of NAO to 2.30±0.15 from 1.38±0.14 and that of Cyt c to 14.20±0.39 from 9.49±0.31.[Conclusion] Mitochondrial dysfunction could be induced by early exposure to lead acetate in HK-2 cells.And then,oxidative damage of diphosphatidyl glycerol would be enhanced,and the release of Cyt c from mitochondria could be induced.Finally,structural damage of mitochondria could be observed in HK-2 cells.更多还原