不同粒径纳米二氧化钛气管暴露对大鼠氧化应激和DNA损伤作用的研究

发布时间：2019-07-04 17:52

【摘要】：目的：纳米材料因其在电学、热学及光学等方面的独特性能，广泛用于医药、化工、计算机、半导体、光学、环境保护、化妆品等许多领域。纳米二氧化钛(Nanoparticle titanium dioxide，Nano-TiO_2)是纳米材料的一种，作为新型光催化剂广泛应用于环境污染物的降解和处理。纳米二氧化钛生产和使用过程中可以进入环境，对环境和生物体产生潜在风险；因此，研究纳米材料对机体产生的健康损害，并探讨其可能的机理就具有十分重要的意义。本文拟通过非暴露式气管注入方法，模拟呼吸道吸收途径，探讨不同粒径纳米二氧化钛对大鼠氧化应激和DNA的损伤作用，为纳米TiO_2的毒理学评价及安全应用提供参考资料。方法： 136只健康Wistar雄性大鼠，随机分为三组，每组12只。实验组采用非暴露式气管注入法进行染毒，分别给予50nm和120nmTiO_2，剂量为2g/kg体重，对照组给予等体积的蒸馏水。观察一周后处死动物取材并进行相应项目的检测。 2大鼠体内Ti含量测定：利用微波消解-电感耦合等离子体质谱（ICP-MS）法检测Nano-TiO_2染毒后大鼠肝脏、肾脏、肺和皮层组织内的Ti含量。 3组织病理学观察：制作肝脏、肾脏、肺和海马组织病理切片，光镜下观察Nano-TiO_2染毒后脏器的病理变化。 4DNA损伤的测定：运用单细胞凝胶电泳试验(SCGE)，检测Nano-TiO_2染毒后，大鼠肝脏、肾脏、肺、皮层、海马组织细胞OTM值的变化。 5细胞氧化损伤指标的测定：利用试剂盒检测Nano-TiO_2染毒后，大鼠肝脏、肾脏、肺、皮层和海马组织中MDA含量以及SOD、GSH-Px活力的变化。 6细胞内活性氧水平的测定：二氢二氯荧光黄双乙酸钠(DCFH-DA)作为荧光探针，采用流式细胞检测技术检测Nano-TiO_2染毒后，大鼠肝脏、肾脏、肺、皮层、海马组织细胞内的活性氧（ROS）水平。 7GADD45α基因mRNA表达的测定：采用聚合酶链式反应(PCR)方法，检测Nano-TiO_2染毒后，大鼠肝脏、肾脏、肺、皮层、海马组织细胞内DNA损伤诱生蛋白45α（GADD45α）基因mRNA表达水平的变化。结果： 1Nano-TiO_2对大鼠的一般状况的影响各组大鼠全部存活。染毒当日、次日，对照组与染毒组均出现饮水、进食量减少，行动慢、活动少，第三日恢复正常，其余未见异常改变。体重正常上升，染毒组与对照组无显著差别。 2Nano-TiO_2对大鼠各组织中Ti含量的影响纳米二氧化钛染毒后，大鼠肝、肾、肺、大脑皮层各组织中Ti含量均有显著增高(P 0.05)。50nmTiO_2染毒后，皮层组织中Ti含量显著高于120nm TiO_2染毒组(P 0.05)；肝脏、肾脏、肺组织中Ti含量虽有所升高，但无统计学意义。 3Nano-TiO_2染毒后大鼠各组织的病理变化纳米二氧化钛染毒后，大鼠肝脏、肾脏、肺和海马组织均出现不同程度的病理变化。染毒组大鼠肝组织出现细胞排列紊乱、水肿，细胞体积增大，细胞质呈空泡状，并有炎性细胞出现，部分区域出现成群片状坏死；肾脏出现肾小管水肿，肾小囊增大，并有片状坏死，肾小球萎缩，间隙增大等变化；肺组织出现肺泡隔增厚，结缔组织增多；海马组织出现细胞核收缩变形，呈不规则形状，异染色质多、常染色质少，三角形嗜酸性粒细胞增多等变化。上述组织的病理变化程度，在50nmTiO_2染毒组较120nmTiO_2染毒组严重。 4Nano-TiO_2对大鼠DNA损伤的影响与对照组相比，大鼠肝脏、肾脏、肺、皮层和海马细胞的OTM均显著增加，(P 0.05)。50nmTiO_2染毒后，肾脏、海马细胞的OTM值显著高于120nm TiO_2染毒组(P 0.05)；肝脏、肺、皮层细胞的OTM值有所增加，但无统计学意义。 5Nano-TiO_2对大鼠氧化应激水平的影响 5.1Nano-TiO_2对大鼠组织细胞内ROS水平的影响与对照组相比，大鼠肝脏、肾脏、肺、皮层和海马细胞中ROS水平均显著升高(P 0.05)。50nmTiO_2染毒后，肾脏、海马细胞的ROS水平显著高于120nm TiO_2染毒组(P 0.05)；肝脏、肺、皮层细胞的ROS水平虽有所增加，但无统计学意义。 5.2Nano-TiO_2对大鼠组织细胞中MDA含量和SOD、GSH-Px活性的影响与对照组相比，大鼠肝脏中MDA含量明显升高，SOD、GSH-Px活性均显著降低(P 0.05)。50nmTiO_2染毒后，MDA含量显著高于120nmTiO_2染毒组(P 0.05)，SOD、GSH-Px活性有所下降，但无统计学意义。与对照组相比，大鼠肾脏中MDA含量明显升高，SOD、GSH-Px活性均显著降低(P 0.05)。50nmTiO_2染毒后，GSH-Px活性显著低于120nmTiO_2染毒组(P 0.05)，SOD活性有所下降但无统计学意义，而MDA含量未见显著改变。与对照组相比，大鼠肺组织中MDA含量明显升高，SOD、GSH-Px活性均显著降低(P 0.05)。50nmTiO_2染毒后，MDA含量显著高于120nmTiO_2染毒组(P 0.05)，SOD、GSH-Px活性有所下降，但无统计学意义。与对照组相比，大鼠皮层中MDA含量明显升高，SOD、GSH-Px活性均显著降低(P 0.05)。50nmTiO_2染毒组较120nm TiO_2染毒组上述改变无统计学意义。与对照组相比，大鼠海马中MDA含量明显升高，SOD、GSH-Px活性均显著降低(P 0.05)。50nmTiO_2染毒后，MDA含量显著高于120nmTiO_2染毒组(P 0.05)，SOD、GSH-Px活性有所下降，但无统计学意义。 6Nano-TiO_2对大鼠组织中GADD45α基因mRNA表达水平的影响与对照组相比，，大鼠肝脏、肾脏、肺、皮层和海马细胞中GADD45α基因mRNA表达水平均显著升高(P 0.05)。50nmTiO_2染毒与120nm TiO_2染毒组之间未见显著差异。结论: 1纳米二氧化钛染毒后，Ti元素可分布于大鼠肝、肾、肺、脑等脏器中；并使这些脏器出现不同程度的病理损害。 2纳米二氧化钛染毒后，可使大鼠各脏器细胞发生DNA损伤；各脏器发生氧化应激，出现氧化损伤；并可大鼠GADD45α基因mRNA表达水平升高。 3随着Nano-TiO_2粒径的减小，对大鼠的损害作用有增加的趋势，但Nano-TiO_2粒径与其损害作用之间的关系，需进一步深入研究。
[Abstract]:Objective: The nanometer material is widely used in many fields such as medicine, chemical industry, computer, semiconductor, optical, environmental protection and cosmetics because of its unique performance in electrical, thermal and optical. Nanosized titanium dioxide (Nano-TiO _ 2) is a kind of nano-material, which is widely used as a new type of photocatalyst for the degradation and treatment of environmental pollutants. In the process of production and use of nano-titanium dioxide, the environment can be entered, and the potential risk to the environment and the organism can be generated; therefore, the health damage of the nano-material to the body can be studied, and the possible mechanism of the nano-titanium dioxide is of great significance. In this paper, the effects of nano-TiO _ 2 on oxidative stress and DNA damage in rats were studied by means of non-exposed air-tube injection method, and reference was made to the toxicological evaluation and safety application of nano-TiO _ 2. square Methods:136 healthy Wistar rats were randomly divided into three groups. 12. The experimental group was treated with the non-exposed tracheal injection method, and the dose was 50 nm and 120 nm TiO _ 2 respectively. The dosage was 2 g/ kg body weight and the control group was given equal volume. distilled water. The animals were sacrificed after one week and the corresponding projects were carried out The content of Ti in rats was determined by microwave digestion-inductively coupled plasma mass spectrometry (ICP-MS). The liver, kidney, lung and cortical tissues of rats after exposure to nano-TiO _ 2 were detected by ICP-MS. The results were as follows: The pathological sections of liver, kidney, lung and hippocampus were made, and the nano-TiO _ 2 was observed under light microscope. The pathological changes of the organs: The determination of DNA damage: The single cell gel electrophoresis (SCGE) was used to test the liver, kidney, lung, cortex and hippocampus of rats. Changes of the value of the OTM in the cells. Determination of the oxidative damage index of the cells: The content of MDA and SOD in the liver, the kidney, the lung, the cortex and the hippocampus of the rats were detected by using the kit. Changes of the activity of GSH-Px. Determination of the level of active oxygen in 6 cells: Dihydrodichlorofluorescein sodium diacetate (DCFH-DA) was used as a fluorescent probe. The liver, kidney, lung, cortex and hippocampus of rats were detected by flow cytometry. Determination of the level of reactive oxygen species (ROS) in the cell. The expression of the 7 GADD45 gene mRNA was determined by the polymerase chain reaction (PCR) method. The DNA damage induced by the DNA in the liver, the kidney, the lung, the cortex and the hippocampus of the rat was detected by the polymerase chain reaction (PCR) method. The expression of the DNA in the rat's liver, kidney, lung, cortex and hippocampus was 45% (GADD45). () basis Changes in mRNA expression level. Results:1 N Nano-TiO _ 2 The normal condition of the rats was affected in all groups. The following day, the next day, the control group and the exposed group had drinking water, the food consumption was reduced, and the operation was slow , the activity is small, the third day is normal, and the rest is not changed The body weight was increased normally, and there was no significant difference between the exposure group and the control group. The content of Ti in the tissues of rat's liver, kidney, lung and cerebral cortex increased significantly after exposure to nano-sized titanium dioxide (P.05). 20 nm TiO _ 2 exposure group (P 0.05); liver The content of Ti in the kidney and lung tissue was increased, but there was no statistical significance. The pathological changes of nano-titanium dioxide in various tissues of the rats after exposure to three nano-TiO _ 2 The liver, the kidney, the lung and the hippocampus of the rats showed different degree of pathological changes. In the group of the rats, the liver tissues of the rats had the cell arrangement disorder, the edema, the volume of the cells increased, the cytoplasm was vacuous, and the inflammatory cells appeared, and the partial region appeared in groups of sheet-like necrosis. The renal tubular edema in the kidney, the increase of the renal capsule, and the changes of the sheet-like necrosis, the atrophy of the glomerulus, the increase of the gap, the increase of the alveolar septum and the increase of the connective tissue in the lung tissue, the deformation of the nucleus in the hippocampus of the hippocampus, and the irregular shape. The changes of the shape, heterochromatin, euchromatin, and the increase of the number of eosinophils in the triangle. The degree of pathological change of the above-mentioned tissues, 120 nm TiO _ 2 in 50 nm TiO _ 2 exposure group The effect of 4 nano-TiO _ 2 on DNA damage in rats was significantly increased in the liver, kidney, lung, cortex and hippocampus of rats (P 0.05). The OTM value was significantly higher than that of the 120 nm TiO _ 2 exposure group ( P 0.05); the OTM values of the liver, the lung, and the cortical cells Increased, but no statistical significance.5 Nano-TiO _ 2 oxidation of rats The effect of the stress level of 5.1 Nano-TiO _ 2 on the level of ROS in rat tissue cells was compared with that of the control group, and the liver, kidney, lung and cortex of the rat The levels of ROS in the cells of the hippocampus and the hippocampus were increased significantly (P 0.05). The ROS level of the cells in the kidney and the hippocampus was significantly higher than that of the 120 nm TiO _ 2 after the 50 nm TiO _ 2 exposure. The level of ROS in the liver, the lung and the cortical cells increased, but there was no statistical significance. 5.2 The effects of nano-TiO _ 2 on the content of MDA and the activity of SOD and GSH-Px in the tissue of the rat were significantly lower than that of the control group (P 0.05), and the activity of SOD and GSH-Px decreased significantly (P 0.05). The MDA content and the activity of SOD and GSH-Px in the liver of the rat were significantly lower than those in the control group (P 0.05). The content is significantly higher than 12 The activity of SOD and GSH-Px in the kidney of the rats was significantly lower than that of the control group (P 0.05). The activity of GSH-Px was significantly lower than that of the control group (P 0.05). The activity of GSH-Px was significantly lower than that of the control group (P 0.05). Compared with the control group, the content of MDA in the lung tissue of the rats increased significantly, and the activity of SOD and GSH-Px decreased significantly (P 0.05). The content of MDA was significant after 50 nm TiO _ 2 exposure. Compared with the control group, the content of MDA in the cortex of the rat was significantly higher, and the activity of SOD and GSH-Px was higher than that of the control group. Compared with the control group, the content of MDA in the hippocampus of the rats was significantly increased, and the activity of SOD and GSH-Px decreased significantly (P 0.05). The MDA content in the hippocampus of the rats was significantly lower than that of the control group (P 0.05). The MDA content in the hippocampus of the rats was significantly lower than that of the control group (P 0.05). The MDA content in the hippocampus of the rats was significantly lower than that of the control group (P 0.05). The MDA content in the hippocampus of the rats was significantly lower than that of the control group (P 0.05). The content of SOD and GSH-Px was higher than that of 120 nm TiO _ 2 exposure group (P 0.05). The effect of 6 nano-TiO _ 2 on the expression level of GADD45 gene mRNA in the rat's tissues was lower than that of the control group, and the GADD45 antigen-base in the liver, kidney, lung, cortex and hippocampus of the rat 鍥爉R There was no significant difference in the level of NA expression (P 0.05). There was no significant difference between the 50 nm TiO _ 2 exposure and the 120 nm TiO _ 2 exposure group. Conclusion: The Ti element can be distributed in the organs of the liver, the kidney, the lung, the brain and other organs of the rat after the nano-titanium dioxide has been exposed. After exposure, DNA damage could occur in various organs of the rat, oxidative stress and oxidative damage were observed in each organ, and the expression level of GADD45 gene mRNA in the rat was increased.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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