【作者】 王军明；

【导师】 蔡原；

【作者基本信息】 中国医科大学 ， 卫生毒理学， 2005， 硕士

【摘要】 前言 铝（Al）是一种低毒元素,在体内大量蓄积时可产生多种毒作用,神经组织尤为敏感,易导致神经元损伤而造成学习和记忆方面的缺陷,因此已日益引起人们的高度关注。 铝的神经毒性机制目前仍不清楚。研究表明铝能促进活性氧（ROS）的产生,而ROS易致脑组织发生脂质过氧化反应。同时,ROS又是细胞凋亡的介质。在一些神经退行性疾病中,铝致细胞凋亡所引起的神经细胞非正常死亡是一个关键机制,而自由基的细胞损伤效应可能是细胞凋亡发生的原因之一。海马是铝作用的主要部位,因此,铝使海马发生细胞凋亡亦可能是这些疾病发生的重要原因。 研究表明,铝对婴幼儿可能特别危险。因此,建立相应的动物模型,观察从大鼠幼年期染铝对脑组织脂质过氧化及海马细胞凋亡的影响对于全面了解铝的神经毒性具有重要意义。本研究采用整体动物实验的方法,通过对刚断乳大鼠进行亚慢性染铝来观察铝对脑组织脂质过氧化和海马细胞凋亡的影响,以明确不同剂量的铝对发育中的中枢神经系统的影响,为阐明铝的神经毒性机制提供理论依据和实验资料。 材料与方法 1.动物分组及染毒 刚断乳Wistar大鼠48只按体重随机分为4组,每组12只,雌雄各半。所设各组分别为:对照组（给予蒸馏水）、低剂量组（给予0.2%AlCl₃水溶液）、中剂量组（给予0.4%AlCl₃水溶液）和高剂量组（给予1.0%AlCl₃水溶液）。实验各组动物通过自由饮水摄入AlCl₃,连续染毒12w。动物室温度18～23℃,相对湿度45～55%。更多还原

【Abstract】 PrefaceAluminum is a chemical element with low toxicity which may produce a variety of toxic effects on human being when it is accumulated in body . Nerve system is especially sensitive to its toxicity and it can cause learning and memory defect. So the neurotoxicity of aluminum has gradually been given more and more concerns.The mechanism of aluminum - induced neurotoxicity is not clear. Aluminum was reported to produce excessive ROS and induce lipid peroxidation in brain. As a medium, ROS may induce apoptosis . Apoptosis induced by aluminum is a key mechanism causing neurons to abnormal death in some neurodegenerative disorders ,and the effect of ROS on neurons may be one of the reasons causing apoptosis. Hippocampus is a major target to aluminum. Therefore, aluminum - induced apoptosis in hippocampus may be an important reason that causes these diseases.Some reports suggested the infants might be at particular risk from Al. To better understand the relationship among Al, LPO in brain and apoptosis in hippocampus after administrated Al to young rats , establishing an animal model has important significance to learn the neurotoxicity of Al entirely. In our present study, we observed LPO in brain and apoptosis in hippocampus after ablactated rats were treated with Al 12w to understand the effect of different dose of Al on developing CNS. The aim is to offer some experimental data to clear the neuro-toxic mechanism of AlMaterial and Methods1. Animal and treatmentAblactated wistar rats, weighting between 50 ~70g,24 males and 24 females , were divided randomly into four groups; 12 of each group ,6 males and 6 females. Their average body weights are almost equal. (1) Control group ( Received distilled water) ; (2) low dose group ( Recived distilled water containing 0.2% AlCl3) ; (3) Medium dose group ( Received distilled water containing 0. 4% AlCl3) ; (4) High dose group ( Received distilled water containing 1. 0% AlCl3) ; The time exposed to Al is 12w . The room temperature is 18 ~ 23℃ , relative humidity is 45 -55% 。2. Methods2. 1 Brain coefficientsBrain coefficients = [ Brain weight( g)/100g B. W]2. 2 Al concentration analysisAl concentration in blood was determined by atomic absorption spectropho-tomery( AAS).2. 3 LPO in Brain analysis2. 3.1 Product of lipid peroxidation ( LPO) was measured by the method of thiobarbituric acid reactive substance ( TBARS).2. 3. 2 Antioxidant substance glutathione (GSH) was estimated by the method of DTNB.2.3.3 The activity of SOD in brain was measured by the modified nitrite method.2. 3.4 The activity of GSH - Px in brain was measured by modified method of DTNB2. 3. 5 Protein was measured by Lowry’s method2.4 Evaluation of apoptic parameters in hippocampus Fas and Bcl -2 protein expression was estimated by an immunohistochemical study .2. 5 HistopathologyTissue selected for histopathological examination was fixed in 10% formalin, embedded in formalin, sectioned at 4μm, and routinely stained with HE and Nissle.3. Data analysisAll data were expressed as means ± SD and analyzed using one - way ANO-VA followed by SPSS 11.5 packages. Pairwise comparisons were made by q -test.Results1. Body weights and brain coefficientsThe body weights did not show any significant changes in the treated rats compared with those of the corresponding group of control animals. The brain coefficients in the rats of the medium dose group were decreased significantly with respect to the corresponding group of control animals.2. Al concentrations in blood analysisThe blood Al concentrations in the rats of all Al treated groups were much higher than those of the rats of control group , and there were significant changes of blood Al concentrations between each 2 groups of 3 Al - treated groups ,too.3. LPO in brainThe MDA levels in brains of all Al - treated groups were significantly increased compared with control group, demonstrating certain dose - effect relation. GSH contents in high dose group were significantly lower than those of control group, low dose group and medium dose group. GSH contents in mediumgroup were also much lower than those in low dose group. The activities of SOD had a declining trend with the enhancement of Al dose . Activities of SOD in high dose group were significantly lower than those in control group, low dose group and medium dose group. The activities of GSH - Px in medium dose group and high dose group were decreased significantly than those in control and low dose groups . The activities in high dose group were also obviously decreased than those in medium dose groups .4. The expression of Bcl -2 protein in hippocampusAlong with the increasing dose of Al, the expression of Bcl 2 protein was decreased progressively. The contents of Bcl - 2 in all Al - treated groups had significantly decreased compared with control group. At the same time, the differences between each 2 groups among 3 Al - treated groups were significant ,too.5. The expression of Fas protein in hippocampusContrast to the expression of Bcl - 2 protein, the expression of Fas protein was increased gradually along with the increasing dose of Al. The expressions of Fas protein in hippocampus in all Al - treated groups were significantly higher than those in control group. There was an increasing trend between low and medium dose group, but no significant difference, which existed between each 2 Al - treated groups of the rest .6. Histopathology of hippocampusThere were no differences among all groups through HE and Nissle stain in our study.DiscussionAl is a chronic accumulating poison, which can impair neurons. Some researchers suggested it can promote the production of ROS and lead to oxidativestress in brain. Several studies had shown that exposure to Al can result in the increase of LPO level , accompanying with the decrease of activities of antioxidant enzymes and contents of antioxidant substances. In our present study ,we observed the increase of LPO level and decrease of ability of antioxidation in brain after 12w treated with Al to ablactated rats. The MDA levels were increased in brain and the activities of SOD were decreased . The levels of GSH and activities of GSH — PX were increased a little in low dose group compared with those of control group. But both of them were decreased remarkably along with the increasing dose of Al. The reason may exist in the stimulating effect of increased LPO in body, causing them increase correspondingly. When the level of LPO exceeds the ability of the body , the contents of GSH and the activities of GSH - Px were decreased intensively . Al can disturb the metabolism of some trace elements such as Cu、Zn、Mn and Se, decreasing their contents in body. Cu、Zn and Mn are necessary components of SOD. When their contents were declined, the activity of SOD may be decreased correspondingly. Lack of Se may decrease the activity of GSH - Px because it is a component of the active center of GSH - Px.Apoptosis, an active process that follows an intrinsic cellular program, and has important physiological implication in the developing nerve system. But on the other hand, apoptosis can also be triggered by drugs and toxins. Al can induce oxidative injury and cause to produce excessive ROS. ROS can mediate excessive apoptosis. Researches had demonstrated that Al can increase the level of LPO in body and induce apoptosis in hippocampus.The proto - oncogene bcl - 2 is rather unique in its ability to block cell death . Bcl - 2 protein has an ability to suppress LPO and protect cell from oxidative stress. In cell culture system someone observed the decreased expression of Bcl - 2 protein in the cortex neurons of rats after treated with AlCl3. No documents have been found the effect of Al on apoptosis in hippocampus of ablactated rats. Our study found that the expression of Bcl -2 protein was decreased signif-更多还原