纳米羟基磷灰石通过溶酶体⁃线粒体通路诱导肾小管上皮细胞的凋亡机制

Mechanism of apoptosis of renal tubular epithelial cells induced by nano⁃hydroxyapatite via lysosomal⁃mito⁃chondrial pathway

目的探索纳米羟基磷灰石(HAP)对肾小管上皮细胞(HK-2细胞)凋亡的影响及其机制.方法将不同浓度的(0、5、10、20、40、80μg/mL)的HAP作用于HK-2细胞,使用MTT、活细胞/死细胞染色检测HAP对HK-2细胞增殖的影响.以0、10、20、40μg/mL的HAP作用HK-2细胞6、24 h,用流式细胞仪检测细胞的凋亡及坏死情况.HAP(40μg/mL)作用于HK-2细胞6 h,用激光共聚焦显微镜观察HAP在细胞内的分布.以0、10、20、40μg/mL的HAP作用HK-2细胞6 h,用酶标仪检测过氧化氢(H2O2)水平和加入抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)后HK-2细胞活性的改变,用流式细胞仪检测活性氧(ROS)、线粒体膜电位(Δψm)、caspase-3水平.结果HAP可明显的抑制HK-2细胞的增殖,且呈浓度-时间依赖性.HAP诱导细胞内ROS升高、线粒体膜电位(Δψm)降低、溶酶体完整性破坏、caspase-3水平增加,从而导致细胞凋亡.结论HAP通过溶酶体和线粒体途径诱导HK-2细胞凋亡,并呈浓度依赖性.

Objective To explore the effect of HAP on the apoptosis of renal tubular epithelial cells(HK⁃2 cells)and its mechanism.Methods HAP at different concentrations(0,5,10,20,40,80μg/mL)was applied to HK⁃2 cells.MTT and live cell/dead cellstaining were used to detect the effect of HAP on HK⁃2 cell proliferation influences.HK⁃2 cellswere treated with HAP at 0,10,20,and 40μg/mL for 6 hours and 24 hours,and the apopto⁃sis and necrosis of the cells were detected by flow cytometry.HAP(40μg/mL)was applied to HK⁃2 cells for 6 hours,and the distribution of HAP in the cells was observed with a laser confocal microscope.HK⁃2 cells were treat⁃ed with Nano⁃HAP at 0,10,20,and 40μg/mL for 6 hours,and the level of hydrogen peroxide(H2O2)was detected with a microplate reader.The antioxidant N⁃acetylcysteine(NAC)After changes in HK⁃2 cell viability,and the lev⁃els of reactive oxygen species(ROS),mitochondrial membrane potential(Δψm),and caspase⁃3 were measured by flow cytometry.Results HAP can significantly inhibit the proliferation of HK⁃2 cells in a concentration⁃time depen⁃dent manner.HAP induces increased intracellular ROS,decreased mitochondrial membrane potential(Δψm),dam⁃aged lysosomal integrity,and increased caspase⁃3 levels,leading to apoptosis.Conclusions HAP induces apopto⁃sis of HK⁃2 cells through lysosomal and mitochondrial pathways in a concentration⁃dependent manner.