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[目的]观察纳米二氧化钛(Nano-TiO2)颗粒诱导细胞内活性氧(ROS)的产生情况。[方法]采用直径5nm锐钛型Nano-TiO2颗粒和直径<10nm、长度40nm金红石型Nano-TiO2颗粒为研究材料,选择细胞质和线粒体部位ROS特异荧光探针二氯荧光素二乙酸酯(DCFH-DA)及双氢罗丹明123(DHR123),用流式细胞仪测定10、20、40μg/mL不同晶型和粒径的Nano-TiO2颗粒在人肺Ⅱ型上皮(A549)细胞作用不同时间所产生的荧光强度。并在5～160μg/mL浓度下,用中性红方法测定两种Nano-TiO2颗粒的细胞毒性。[结果]锐钛型Nano-TiO2颗粒和金红石型Nano-TiO2颗粒诱导细胞质和线粒体内ROS结果基本相同,作用5min就可检测到ROS,一直持续到2h,而高剂量组诱导ROS大于低剂量组。锐钛型Nano-TiO2颗粒产生ROS峰值在30min,而金红石型Nano-TiO2颗粒产生ROS峰值在60min。用流式细胞仪检测ROS实验中意外出现纳米颗粒峰,峰值高低与纳米颗粒诱导ROS荧光强度反应一致,表明ROS反应与染毒剂量成正比关系,可在同一检测中得到证明。而在实验中未发现两种Nano-TiO2颗粒存在细胞毒性。[结论]Nano-TiO2颗粒可以诱导细胞质和线粒体内ROS产生。
[Objective] To observe the production of intracellular reactive oxygen species (ROS) induced by Nano-TiO2 particles. [Method] Nano-TiO2 particles with diameter of 5 nm and rutile Nano-TiO2 particles with diameter of <10 nm and length of 40 nm were used as materials to select the ROS-specific fluorescent probe of cytoplasm and mitochondria. Dichlorofluorescein diacetate (DCFH -DA) and DHR123 (DHR123). Flow cytometry was used to determine the effect of 10,20,40μg / mL Nano-TiO2 particles with different crystal forms and particle sizes on the proliferation of human lung epithelial type Ⅱ (A549) cells at different times The resulting fluorescence intensity. The cytotoxicities of two kinds of Nano-TiO2 particles were measured by the neutral red method at the concentration of 5 ~ 160μg / mL. [Results] The results of anatase-type Nano-TiO2 particles and rutile-type Nano-TiO2 particles induced similar ROS results in cytoplasm and mitochondria. ROS was detected after 5 minutes and continued until 2 hours, while ROS induced by high dose group was higher than that of low dose group . The anatase Nano-TiO2 particles produced ROS peak at 30min, while the rutile Nano-TiO2 particles produced ROS peak at 60min. The results of flow cytometry showed that there was an unexpected peak of ROS in ROS. The peak value was consistent with the fluorescence intensity of ROS induced by nanoparticles. The results showed that the ROS response was proportional to the dose of ROS, which could be proved by the same assay. No cytotoxicity was observed in the two Nano-TiO2 particles in the experiment. [Conclusion] Nano-TiO2 particles can induce ROS production in cytoplasm and mitochondria.