纳米二氧化钛颗粒诱导人肺Ⅱ型上皮细胞活性氧的研究
A Study on Induction of the Reactive Oxygen Species (ROS)in A549 Cells by Titanium Dioxide Nanoparticles
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摘要:
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。 Abstract:Objective To observe dynamically the induction of reactive oxygen species (ROS) by titanium dioxide nanoparticles in the human epithelial cell line A549, which originated from a human lung carcinoma and composed of polarized alveolar type Ⅱ cells. Methods The A549 cells were exposed to 10, 20, 40 μg/mL 5 nm diameter anatase TiO2 particles and <10nm(diameter)& #215;40nm(length)rutile TiO2 particles. Flow cytometry was used to analyze the fluorescence probes for ROS in the cells, consisting of Diehlo (DCFH-DA)located predominantly in cytosol, and Dihydrorhodamine 123 (DHR123)as mitochondriaassociated probe. Results The biological response in cytoplasm and mitochondria to release ROS induced by the two types of TiO2 particles was similar. ROS could be detected as rapid as in 5 minutes after the exposure and could sustain for 120 min under tested concentrations (5-160μg/mL)and with dose dependent response. The peak of ROS was generated in 30 rain after 5 nm diameter anatase TiO2 particles treatment and 60 rain after <10nm(diameter)& #215;40 nm(length)rutile TiO2 particles treatment. Flow cytometric analysis showed that nanoparticles unexpectedly appeared in determination of ROS each time, and the stronger ROS fluorescence intensity the higher peak of nanoparticles could be observed. This result suggested that it might be possible to use a simple and easy method of flow cytometry to evaluate uptake potential of nanoparticles in mammalian cells and to replace transmission electron microscope (TEM) for quantifying nanoparticles in biological specimens. Conclusion 5 nm diameter anatase and <10 nm (diameter)& #215;40 nm(length) ruffle TiO2 particles can induce ROS of cytosol and mitochondria in A549 cells.
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