乙酸铅诱发人肾小管上皮细胞线粒体损伤的体外研究
An In Vitro Study on the Mitochondria Damage of Human Kidney Cells Induced by Lead Acetate
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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& #177;0.25下降到400μmol/L组的2.90& #177;0.26或从正常对照组的4.44& #177;0.20下降到12 h组的2.34& #177;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& #177;0.18下降到400 μmol/L组的0.91& #177;0.18或从正常对照组的2.52& #177;0.01下降到24 h组的1.50& #177;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。50 μmol/L甘露醇能使NAO荧光强度从1.38& #177;0.14恢复至2.30& #177;0.15,将Cyt c荧光强度从9.49& #177;0.31恢复至14.20& #177;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。 Abstract:Objective To explore the possible mechanism of mitochondria damage in human kidney cells (HK-2 cells) in duced 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& #177;0.25 in normal control group to 2.90& #177;0.26 in 400 μmol/L exposure group and from 4.44& #177;0.20 in normal control group to 2.34& #177;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 timedependent manner, significantly down from 2.45& #177;0.18 in normal control group to 0.91& #177;0.18 in 400 μmol/L exposure group and from 2.52& #177;0.01 in normal control to 1.50& #177;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& #177;0.15 from 1.38& #177;0.14 and that of Cyt c to 14.20& #177;0.39 from 9.49& #177;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.
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