不同粒徑納米二氧化鈦氣管暴露對大鼠氧化應激和DNA損傷作用的研究
發(fā)布時間：2019-07-04 17:52
【摘要】：目的：納米材料因其在電學、熱學及光學等方面的獨特性能，廣泛用于醫(yī)藥、化工、計算機、半導體、光學、環(huán)境保護、化妝品等許多領域。納米二氧化鈦(Nanoparticle titanium dioxide，Nano-TiO_2)是納米材料的一種，作為新型光催化劑廣泛應用于環(huán)境污染物的降解和處理。納米二氧化鈦生產(chǎn)和使用過程中可以進入環(huán)境，對環(huán)境和生物體產(chǎn)生潛在風險；因此，研究納米材料對機體產(chǎn)生的健康損害，并探討其可能的機理就具有十分重要的意義。本文擬通過非暴露式氣管注入方法，模擬呼吸道吸收途徑，探討不同粒徑納米二氧化鈦對大鼠氧化應激和DNA的損傷作用，為納米TiO_2的毒理學評價及安全應用提供參考資料。 方法： 136只健康Wistar雄性大鼠，隨機分為三組，每組12只。實驗組采用非暴露式氣管注入法進行染毒，分別給予50nm和120nmTiO_2，劑量為2g/kg體重，對照組給予等體積的蒸餾水。觀察一周后處死動物取材并進行相應項目的檢測。 2大鼠體內(nèi)Ti含量測定：利用微波消解-電感耦合等離子體質(zhì)譜（ICP-MS）法檢測Nano-TiO_2染毒后大鼠肝臟、腎臟、肺和皮層組織內(nèi)的Ti含量。 3組織病理學觀察：制作肝臟、腎臟、肺和海馬組織病理切片，光鏡下觀察Nano-TiO_2染毒后臟器的病理變化。 4DNA損傷的測定：運用單細胞凝膠電泳試驗(SCGE)，檢測Nano-TiO_2染毒后，大鼠肝臟、腎臟、肺、皮層、海馬組織細胞OTM值的變化。 5細胞氧化損傷指標的測定：利用試劑盒檢測Nano-TiO_2染毒后，大鼠肝臟、腎臟、肺、皮層和海馬組織中MDA含量以及SOD、GSH-Px活力的變化。 6細胞內(nèi)活性氧水平的測定：二氫二氯熒光黃雙乙酸鈉(DCFH-DA)作為熒光探針，采用流式細胞檢測技術檢測Nano-TiO_2染毒后，大鼠肝臟、腎臟、肺、皮層、海馬組織細胞內(nèi)的活性氧（ROS）水平。 7GADD45α基因mRNA表達的測定：采用聚合酶鏈式反應(PCR)方法，檢測Nano-TiO_2染毒后，大鼠肝臟、腎臟、肺、皮層、海馬組織細胞內(nèi)DNA損傷誘生蛋白45α（GADD45α）基因mRNA表達水平的變化。 結(jié)果： 1Nano-TiO_2對大鼠的一般狀況的影響 各組大鼠全部存活。染毒當日、次日，對照組與染毒組均出現(xiàn)飲水、進食量減少，行動慢、活動少，第三日恢復正常，其余未見異常改變。體重正常上升，染毒組與對照組無顯著差別。 2Nano-TiO_2對大鼠各組織中Ti含量的影響 納米二氧化鈦染毒后，大鼠肝、腎、肺、大腦皮層各組織中Ti含量均有顯著增高(P 0.05)。50nmTiO_2染毒后，皮層組織中Ti含量顯著高于120nm TiO_2染毒組(P 0.05)；肝臟、腎臟、肺組織中Ti含量雖有所升高，但無統(tǒng)計學意義。 3Nano-TiO_2染毒后大鼠各組織的病理變化 納米二氧化鈦染毒后，大鼠肝臟、腎臟、肺和海馬組織均出現(xiàn)不同程度的病理變化。染毒組大鼠肝組織出現(xiàn)細胞排列紊亂、水腫，細胞體積增大，細胞質(zhì)呈空泡狀，并有炎性細胞出現(xiàn)，部分區(qū)域出現(xiàn)成群片狀壞死；腎臟出現(xiàn)腎小管水腫，腎小囊增大，并有片狀壞死，腎小球萎縮，間隙增大等變化；肺組織出現(xiàn)肺泡隔增厚，結(jié)締組織增多；海馬組織出現(xiàn)細胞核收縮變形，呈不規(guī)則形狀，異染色質(zhì)多、常染色質(zhì)少，三角形嗜酸性粒細胞增多等變化。上述組織的病理變化程度，在50nmTiO_2染毒組較120nmTiO_2染毒組嚴重。 4Nano-TiO_2對大鼠DNA損傷的影響 與對照組相比，大鼠肝臟、腎臟、肺、皮層和海馬細胞的OTM均顯著增加，(P 0.05)。50nmTiO_2染毒后，腎臟、海馬細胞的OTM值顯著高于120nm TiO_2染毒組(P 0.05)；肝臟、肺、皮層細胞的OTM值有所增加，但無統(tǒng)計學意義。 5Nano-TiO_2對大鼠氧化應激水平的影響 5.1Nano-TiO_2對大鼠組織細胞內(nèi)ROS水平的影響 與對照組相比，大鼠肝臟、腎臟、肺、皮層和海馬細胞中ROS水平均顯著升高(P 0.05)。50nmTiO_2染毒后，腎臟、海馬細胞的ROS水平顯著高于120nm TiO_2染毒組(P 0.05)；肝臟、肺、皮層細胞的ROS水平雖有所增加，但無統(tǒng)計學意義。 5.2Nano-TiO_2對大鼠組織細胞中MDA含量和SOD、GSH-Px活性的影響 與對照組相比，大鼠肝臟中MDA含量明顯升高，SOD、GSH-Px活性均顯著降低(P 0.05)。50nmTiO_2染毒后，MDA含量顯著高于120nmTiO_2染毒組(P 0.05)，SOD、GSH-Px活性有所下降，但無統(tǒng)計學意義。 與對照組相比，大鼠腎臟中MDA含量明顯升高，SOD、GSH-Px活性均顯著降低(P 0.05)。50nmTiO_2染毒后，GSH-Px活性顯著低于120nmTiO_2染毒組(P 0.05)，SOD活性有所下降但無統(tǒng)計學意義，而MDA含量未見顯著改變。 與對照組相比，大鼠肺組織中MDA含量明顯升高，SOD、GSH-Px活性均顯著降低(P 0.05)。50nmTiO_2染毒后，MDA含量顯著高于120nmTiO_2染毒組(P 0.05)，SOD、GSH-Px活性有所下降，但無統(tǒng)計學意義。 與對照組相比，大鼠皮層中MDA含量明顯升高，SOD、GSH-Px活性均顯著降低(P 0.05)。50nmTiO_2染毒組較120nm TiO_2染毒組上述改變無統(tǒng)計學意義。 與對照組相比，大鼠海馬中MDA含量明顯升高，SOD、GSH-Px活性均顯著降低(P 0.05)。50nmTiO_2染毒后，MDA含量顯著高于120nmTiO_2染毒組(P 0.05)，SOD、GSH-Px活性有所下降，但無統(tǒng)計學意義。 6Nano-TiO_2對大鼠組織中GADD45α基因mRNA表達水平的影響 與對照組相比，，大鼠肝臟、腎臟、肺、皮層和海馬細胞中GADD45α基因mRNA表達水平均顯著升高(P 0.05)。50nmTiO_2染毒與120nm TiO_2染毒組之間未見顯著差異。 結(jié)論: 1納米二氧化鈦染毒后，Ti元素可分布于大鼠肝、腎、肺、腦等臟器中；并使這些臟器出現(xiàn)不同程度的病理損害。 2納米二氧化鈦染毒后，可使大鼠各臟器細胞發(fā)生DNA損傷；各臟器發(fā)生氧化應激，出現(xiàn)氧化損傷；并可大鼠GADD45α基因mRNA表達水平升高。 3隨著Nano-TiO_2粒徑的減小，對大鼠的損害作用有增加的趨勢，但Nano-TiO_2粒徑與其損害作用之間的關系，需進一步深入研究。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114
本文編號：2510101
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114
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