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[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。
[Objective] To investigate the effect of lead on mitochondrial damage of human renal cells (HK-2) and its possible mechanism. [Method] HK-2 cells, rhodamine 123 (Rh123) and 10-nonyl acridine orange (NAO) combined with fluorescence chemiluminescence apparatus were cultured in different concentrations of lead acetate The levels of mitochondrial membrane potential and cardiolipin were measured. The release of mitochondrial cytochrome c (Cyt c) was detected by immunofluorescence combined with flow cytometry. The effect of lead on the changes of cardiolipin and Cyt c was observed with mannitol. [Result] Lead acetate could reduce the mitochondrial membrane potential in HK-2 cells in a dose-and time-dependent manner. Fluorescence intensity decreased from 4.56 ± 0.25 in normal control group to 2.90 ± 0.26 in 400μmol / L group or 4.44 ± 0.20 from normal control group Compared with the normal control group, the difference was statistically significant (P <0.05). Lead acetate significantly reduced the time and dose-dependent decrease of the fluorescence intensity of NAO in HK-2 cells. The fluorescence intensity decreased from 2.45 ± 0.18 in normal control group to 0.91 ± 0.18 in 400μmol / L group or from 2.52 ± 0.01 to 1.50 ± 0.05 in 24 h group, which was significantly different from that in control group (P <0.05). Lead acetate induced mitochondrial Cyt c release, which reduced the Cyt c fluorescence intensity in a dose-dependent and time-dependent manner. 50μmol / L mannitol could restore the fluorescence intensity of NAO from 1.38 ± 0.14 to 2.30 ± 0.15, restore the fluorescence intensity of Cyt c from 9.49 ± 0.31 to 14.20 ± 0.39, and significantly inhibit the oxidation of lead on cardiolipin and the release of Cyt c. [Conclusion] Lead may collapse through mitochondrial membrane potential in HK-2 cells at early stage, resulting in mitochondrial dysfunction, further enhancing the oxidative damage of mitochondrial cardiolipin and releasing Cyt c, resulting in the structural damage of mitochondria.