纳米氧化钛对大鼠神经胶质细胞的毒性作用

Cytotoxicity of titanium dioxide nanoparticles in rat neuroglia cells

目的研究纳米氧化钛(nano-TiO2)对大鼠神经胶质细胞的毒性作用。方法①体外实验:制备3种粒径10,20和200nm的nano-TiO2颗粒悬液,分别以6.25,12.5,25,50和100mg·L-1对大鼠星形胶质细胞进行72h染毒培养,应用磺基罗丹明B法检测nano-TiO2对星形胶质细胞存活率的影响。②体内实验:Wistar大鼠气管内分别注入上述3种粒径的nano-TiO2悬液,每种粒径设0.1,1.0和10.0mg·kg-13个剂量组,每组3只,72h后分别用电感耦合等离子质谱和放射免疫法检测大鼠脑组织中nano-TiO2的含量和白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和IL-10水平的变化,并通过光学显微镜和透射电镜观察nano-TiO2对大鼠神经胶质细胞形态的影响。结果①体外实验发现,nano-TiO2对大鼠星形胶质细胞存活率的抑制作用具有明显的浓度-效应关系,3种粒径10,20和200nm的nano-TiO2与胶质细胞培养72h,其半数抑制浓度分别为55.9,66.0和3827.0mg·L-1;细胞形态亦发生明显变化,细胞排列稀疏,间隙增大,胞内颗粒物增多,细胞透明度下降。②体内实验发现,粒径10和20nm的nano-TiO20.1mg·kg-1及粒径200nm的nano-TiO20.1,1.0和10.0mg·kg-1组,大鼠脑组织中的nano-TiO2,IL-1β,TNF-α及IL-10浓度与对照组比较无明显变化;粒径10和20nm的nano-TiO21.0及10.0mg·kg-1组大鼠脑组织中nano-TiO2,IL-1β,TNF-α及IL-10浓度随nano-TiO2剂量的增加而增加;病理学观察结果表明,nano-TiO2破坏大鼠血脑屏障,造成脑组织坏死,并进入神经胶质细胞内,引起炎症反应和细胞水肿。结论Nano-TiO2对大鼠神经胶质细胞具有细胞毒性作用,其作用强度与粒径大小有关,其作用机制可能与诱导炎症反应有关。

OBJECTIVE To study the cytotoxicity of titanium dioxide nanoparticles (nano-TiO2)in neuroglia cells. METHODS ① In vitro, rat astrocytes were exposed to nano-TiO2 of 3 different diameters (10, 20 and 200 nm) at 5 different concentrations (6.25, 12.5, 25, 50 and 100 mg·L-1). Sulforhodamine B staining assay was carried out to evaluate the survival rate of nano-TiO2-treated cells for 72 h. ② In vivo, nano-TiO2 of these 3 different diameters at 0.1, 1.0 and 10.0 mg·kg-1 were intratracheally instilled into the bronchus of Wistar rats, respectively, 3 rats in each group. Seventy-two hours later, the content of nano-TiO2 and levels of interleukin-1β(IL-1β), tumor necrosis factor-α (TNF-α) and IL-10 in brain tissue were measured with ICP-MS and radioimmunoassays. In addition, cellular morphology of rat neuralgia cells was observed by the light microscope and transmission electron microscope. RESULTS ① In vitro, the inhibiting effect of nano-TiO2 on the cell survival rate was significantly enhanced in a concentration-dependent manner and IC50of nano-TiO2 of the 3 diameters was 55.9, 66.0 and 3827.0 mg·L-1 at 72 h. There were also obvious changes in cellular configuration, such as sparse arrangement, increscent cell space, increased intracellular particles and decreasing cellular transparency. ② In vivo, when the rats were exposed to nano-TiO2 of 10 and 20 nm in diameter in 0.1 mg·kg-1or nano-TiO2 of 200 nm in diameter in 3 dosages, the contents of nano-TiO2, IL-1β,TNF-α and IL-10 in rat brain tissue did not show significant difference in the control and nano-TiO2-treated groups; when the rats were exposed to nano-TiO2 of 10 and 20 nm in diameter in 1.0 and 10.0 mg·kg-1, the contents of nano-TiO2 and the 3 cytokines in brain tissue increased compared with control group, especially nano-TiO2 in 10.0 mg·kg-1 group. Pathological observation indicated that the blood-brain-barrier was destroyed by nano-TiO2, which resulted in brain tissue necrosis and the entrance of nano-TiO2 into neuroglia cells to induce inflammation and cellular edema. CONCLUSION Nano-TiO2 is toxic to rat neuroglia cells. This toxicity may relate to the particle size and the mechanism may be associated with its induction of inflammation.