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目的研究氧化应激在纳米氧化锌引起的人非小细胞肺癌细胞(H1299)细胞自噬和凋亡之间的作用。方法实验分为实验组和对照组,对照组给予正常的培养基而实验组在培养基中分别给予2.5、10和25μg/ml(低,中,高剂量组)浓度的纳米氧化锌。向细胞中转染绿色荧光蛋白-微管相关蛋白1轻链3(GFP-LC3)质粒,通过观察绿色荧光斑点确定自噬的发生。Western blotting检测LC3-Ⅱ来验证绿色荧光斑点确定的细胞自噬。钙黄绿素乙酰甲酯(calcein AM)和碘化丙啶(PI)染色来确定活细胞和晚期凋亡细胞。超氧化物阴离子荧光探针(DHE)染色检测细胞内活性氧(ROS)的聚集。应用动态光散射测定纳米氧化锌在培养基中的分散状态。结果对照组细胞有少量的DHE被氧化成红色,也出现了绿色荧光斑点的聚集,但没有细胞被PI染成红色。低剂量组被氧化的DHE开始增多,绿色荧光斑点开始增多,少量细胞被PI染成红色;中、高剂量组中大量DHE被氧化,绿色荧光斑点显著增多,大量细胞被PI染成红色。实验组纳米氧化锌在培养基中分散良好。结论纳米氧化锌在H1299细胞中引起了氧化应激,氧化应激诱发了细胞的自噬,细胞自噬功能的紊乱诱导了细胞的凋亡。
Objective To investigate the role of oxidative stress in autophagy and apoptosis of human non-small cell lung cancer (H1299) cells induced by nano-ZnO. Methods The experiment was divided into experimental group and control group, the control group was given normal medium and the experimental group were given 2.5, 10 and 25μg / ml (low, medium and high dose) concentration of nano-ZnO in the medium. The green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) plasmid was transfected into the cells, and the occurrence of autophagy was confirmed by observing the green fluorescent spots. LC3-Ⅱ was detected by Western blotting to verify the autophagy determined by green fluorescent spots. Calcein AM and PI staining were used to determine viable cells and late apoptotic cells. Superoxide anion fluorescent probe (DHE) staining was used to detect intracellular reactive oxygen species (ROS) accumulation. Determination of Dispersion of Zinc Oxide in Culture Medium by Dynamic Light Scattering. Results In the control group, a small amount of DHE was oxidized to red, and green fluorescent spot aggregation also occurred. However, no cells were stained with PI. In low dose group, DHE started to increase, green fluorescent spots began to increase, a small number of cells were PI stained red; middle and high dose group was oxidized a large number of DHE, a significant increase in green fluorescent spots, a large number of cells were PI stained red. The experimental group of nano-ZnO dispersed well in the medium. Conclusion Nano-ZnO induced oxidative stress in H1299 cells and oxidative stress induced autophagy. The disorder of cell autophagy induced cell apoptosis.