纳米与普通粒径二氧化钛粉尘体内外的毒性研究

发布时间：2018-06-07 23:56

本文选题：纳米二氧化钛 + 普通粒径二氧化钛　；参考：《宁波大学》2013年硕士论文

【摘要】：目的通过体内动物实验和体外细胞实验，系统研究纳米粒径二氧化钛粉尘（TiO_2NPs）与普通粒径二氧化钛粉尘（TiO_2FPs）对动物和培养细胞的毒性；在此基础上探索了N-乙酰半胱氨酸（NAC）对TiO_2NPs产生细胞毒性的拮抗作用及机制，为TiO_2NPs的职业与环境毒性评价和安全使用提供科学依据。方法体内动物实验：通过采用尾静脉注射法对小鼠进行不同剂量染毒，Horn’s法计算TiO_2NPs与TiO_2FPs的半数致死剂量（LD50）；脏器称重计算脏器系数；全血检测血常规指标；分离血清，检测血生化指标；HE染色法观察组织的病理损伤情况；骨髓细胞微核实验法检测TiO_2NPs是否具有遗传毒性。体外细胞实验：采用FITC标记过的TiO_2NPs染毒小鼠JB6上皮细胞，研究TiO_2NPs在染毒细胞中的荧光定位；采用荧光染料H2DCFDA和DHE染色观察细胞内ROS的产生情况，比较TiO_2NPs与TiO_2FPs对细胞的氧化应激毒性；Hoechst法染色细胞核，比较TiO_2NPs与TiO_2FPs对细胞核的毒性；Luciferase法比较TiO_2NPs与TiO_2FPs对AP-1细胞荧光素酶表达的影响；蛋白免疫印迹法比较TiO_2NPs与TiO_2FPs染毒后对细胞C-jun和P53蛋白表达的影响，探索TiO_2NPs与TiO_2FPs致肿瘤作用的分子机制。抗氧化剂NAC对TiO_2NPs氧化应激毒性的抑制作用：通过在细胞染毒前加入20nM抗氧化剂NAC，检测细胞活性氧自由基的产生变化情况，观察NAC是否可以对TiO_2NPs产生的细胞毒性起到一定的拮抗作用。结果体内实验： LD50结果表明雌性小鼠（948mg/kg）对TiO_2NPs毒性的敏感性大于雄性小鼠（1392mg/kg）。TiO_2NPs染毒后，可不同程度的损伤小鼠的肝、肺和肾，呈一定的剂量反应关系。但是，并未发现TiO_2NPs具有遗传毒性；TiO_2FPs LD50为114.9mg/kg。与TiO_2NPs毒性相似，，也可引起小鼠的肝、肺和肾的毒性损伤。体外实验：通过FITC荧光探针定位，发现TiO_2NPs可以透过细胞膜，未见其侵入细胞核内；Hoechst染色法发现TiO_2NPs造成的细胞凋亡明显大于TiO_2FPs；H2DCFDA，DHA，Hoechst共同染色结果显示，在相同的染毒剂量下，TiO_2NPs所产生的活性氧自由基（ROS）明显大于TiO_2FPs，且存在一定的剂量反应关系；TiO_2NPs与TiO_2FPs均可引起细胞AP-1荧光素酶的表达升高，并且，TiO_2NPs组的升高程度明显高于TiO_2FPs组。此外，蛋白免疫印迹实验结果显示，25μg/cm2的TiO_2NPs可引起C-jun蛋白表达量升高和P53水平下降。抗氧化剂NAC对TiO_2NPs产生毒性的抑制作用：加入抗氧化剂NAC后，不仅细胞产生的ROS受到了抑制，同时细胞AP-1荧光素酶的表达也有所降低。说明NAC对TiO_2NPs产生的细胞氧化应激有一定的拮抗作用。结论体内实验结果表明：TiO_2NPs与TiO_2FPs经过尾静脉染毒可以引起小鼠脏器不同程度的损伤，提示经血液接触一定量的TiO_2NPs与TiO_2FPs均可能对机体造成潜在危害。体外实验结果表明：TiO_2NPs可以通过细胞膜进入细胞浆内，引起促癌基因表达上调，其毒性主要表现为细胞内氧化应激损伤，且毒性大于TiO_2FPs。此外，抗氧化剂NAC可以考虑作为一种新型的药物用来拮抗TiO_2NPs对细胞产生的氧化应激损伤。
[Abstract]:objective
Through in vivo animal experiments and in vitro cell experiments, the toxicity of nanoparticle titanium dioxide dust (TiO_2NPs) and ordinary particle size titanium dioxide dust (TiO_2FPs) on animals and cultured cells was systematically studied. On this basis, the antagonism and mechanism of N- acetylcysteine (NAC) on the toxicity of TiO_2NPs producing cells were explored, which was a TiO_2NPs job. It provides scientific basis for environmental toxicity assessment and safe use.
Method
In vivo animal experiments: the mice were poisoned by the tail vein injection, the Horn 's method was used to calculate the half lethal dose of TiO_2NPs and TiO_2FPs (LD50); the organ weight was weighed to calculate the organ coefficient; the whole blood was used to detect the blood routine index; the serum was separated and the blood biochemical index was detected; the pathological damage of the tissue was observed by HE staining. Bone marrow cell micronucleus test was used to detect whether TiO_2NPs had genetic toxicity. In vitro cell experiment: using FITC labeled TiO_2NPs to poison the JB6 epithelial cells of mice, the fluorescence localization of TiO_2NPs in the infected cells was studied. The production of ROS in the cells was observed by the fluorescent dye H2DCFDA and DHE staining, and the TiO_2NPs and TiO_2FPs were compared. Cytotoxicity of cellular oxidative stress; Hoechst staining of nuclei to compare the toxicity of TiO_2NPs and TiO_2FPs to the nucleus; Luciferase method compared the effect of TiO_2NPs and TiO_2FPs on the expression of luciferase in AP-1 cells; protein immunoblotting was used to compare the effect of TiO_2NPs and TiO_2FPs on the expression of C-jun and P53 proteins after TiO_2NPs and TiO_2FPs. The molecular mechanism of O_2FPs induced tumor action. The inhibitory effect of antioxidant NAC on the oxidative stress toxicity of TiO_2NPs: by adding 20nM antioxidant NAC before the cells were infected, the changes in the production of free radicals of reactive oxygen species were detected, and the effect of NAC on the cytotoxicity of TiO_2NPs produced by TiO_2NPs was observed.
Results in the experiment in vivo: LD50 results showed that the sensitivity of female mice (948mg/kg) to TiO_2NPs toxicity was greater than that of male mice (1392mg/kg).TiO_2NPs, and there was a certain dose response relationship between the liver, lung and kidney of mice in varying degrees. However, TiO_2NPs was not found to have genetic toxicity; TiO_2FPs LD50 was 114.9mg/kg. and TiO_2N. Ps is similar in toxicity and can cause liver, lung and kidney toxicity in mice.
In vitro experiment: it was found that TiO_2NPs could penetrate cell membrane and not intruder into the nucleus by FITC fluorescence probe. The apoptosis of TiO_2NPs caused by Hoechst staining was obviously greater than that of TiO_2FPs; H2DCFDA, DHA, Hoechst CO staining showed that the reactive oxygen free radical (ROS) produced by TiO_2NPs under the same amount of poison. It was obviously greater than TiO_2FPs, and there was a certain dose response relationship. Both TiO_2NPs and TiO_2FPs could cause the increase of AP-1 luciferase expression in cell, and the increase of TiO_2NPs group was significantly higher than that of TiO_2FPs group. In addition, the results of protein immunoblot test showed that the 25 u g/cm2 TiO_2NPs could cause the increase of C-jun protein expression and P53 level. Decrease. Anti oxidant NAC inhibits the toxicity of TiO_2NPs: after adding antioxidant NAC, not only the ROS of the cells is inhibited, but also the expression of AP-1 luciferase is also reduced. It shows that NAC has certain antagonism to the oxidative stress produced by TiO_2NPs.
conclusion
The results of the experiment in vivo show that TiO_2NPs and TiO_2FPs can cause different degrees of damage to the organs of mice through the tail vein, suggesting that the certain amount of TiO_2NPs and TiO_2FPs may cause potential harm to the body. In vitro experimental results show that TiO_2NPs can enter the cytoplasm through the cell membrane and cause the cancer promoting gene table. Up regulation, its toxicity is mainly manifested in intracellular oxidative stress damage, and the toxicity is greater than TiO_2FPs.. Antioxidant NAC can be considered as a new drug used to antagonize oxidative stress damage caused by TiO_2NPs to cells.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【参考文献】