维生素E对镉致小鼠大脑皮质神经元和星形胶质细胞损伤的保护作用

发布时间：2018-07-10 02:53

本文选题：镉 + 神经元　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的探讨维生素E(Vitamin E,VE)对镉致小鼠大脑皮质神经元与星形胶质细胞损伤的保护作用。方法一、体外实验(1)体外培养原代大脑皮质神经元和星形胶质细胞,并选择神经元特异性蛋白(MAP2)、星形胶质细胞特异性蛋白(GFAP)和小胶质细胞特异性蛋白(CD11b)进行细胞纯度鉴定;(2)以不同浓度镉溶液染毒,于倒置相差显微镜下分别观察神经元和星形胶质细胞在不同时间点形态学变化,加入维生素E干预后,观察维生素E对染镉皮质神经元和星形胶质细胞的形态学,分光光度计检测SOD活性、MDA含量,Western blot检测Bcl-2/Bax、caspase-3蛋白表达。二、体内实验(1)健康昆明种雄性小鼠60只随机分为3组,每组20只。慢性镉中毒组(Cd组):氯化镉溶液2mg/kg经腹腔皮下注射,每周两次;慢性镉中毒加维生素E组(Cd+VE组):注射氯化镉染毒,同时每天进行维生素E溶液10 mg/kg灌胃;对照组:注射生理盐水。(2)建模13周后,Y形迷宫检测各组小鼠学习记忆能力,检测大脑皮质组织匀浆SOD活性和MDA含量,尼氏染色观察额叶皮质区神经元尼氏体变化,透射电镜观察大脑额叶皮质神经元超微结构变化,免疫组化检测大脑额叶皮质区GFAP和caspase-3阳性表达细胞,Western blot检测大脑皮质组织caspase-3的蛋白表达。结果(1)体外培养的原代大脑皮质神经元和星形胶质细胞,经免疫荧光鉴定纯度均可达到90%以上;(2)体外实验中,与对照组比较,随着染镉浓度的增加和染镉时间的延长,神经元与星形胶质细胞形态学损伤逐渐加重;加入维生素E干预后,与单纯的染镉组比较,神经元与星形胶质细胞形态学损伤减轻,细胞的SOD活性升高、MDA含量下降(P0.05),Bcl-/Bax比值升高、caspase-3蛋白表达下降(P0.05);VE干预组与对照组比较,各项指标仍有差异(P0.05);(3)在体研究中,Cd组小鼠学习记忆能力明显低于对照组和Cd+VE组(P0.05);Cd组小鼠大脑皮质组织中SOD活性均低于对照组和Cd+VE组、MDA含量均高于对照组和Cd+VE组(P0.05);与对照组和Cd+VE组相比,Cd组大脑皮质额叶神经元尼氏体减少,神经元超微结构损伤严重,GFAP与caspase-3阳性细胞增多,大脑皮质组织caspase-3蛋白表达显著增高(P0.05);与对照组比较,Cd+VE组大脑皮质神经元尼氏体轻度减少,神经元超微结构轻度受损,caspase-3与GFAP阳性细胞高于对照组,皮质组织caspase-3蛋白表达同样高于对照组(P0.05)。结论维生素E对镉致小鼠大脑皮质神经元和星形胶质细胞的损伤具有一定的保护作用,且维生素E的保护作用与其抗氧化、减少细胞凋亡有关。
[Abstract]:Objective to investigate the protective effect of vitamin E (Vitamin E) on the injury of cerebral cortex neurons and astrocytes induced by cadmium in mice. Methods 1. Primary cortical neurons and astrocytes were cultured in vitro. Neuron specific protein (MAP2), astrocyte specific protein (GFAP) and microglia specific protein (CD11b) were selected for cell purity identification. The morphologic changes of neurons and astrocytes at different time points were observed under inverted phase contrast microscope. After vitamin E was added, the morphologic changes of neurons and astrocytes in cadmium-exposed cortex were observed. The content of SOD and MDA was measured by spectrophotometer and the expression of Bcl-2 / Bax-caspase-3 protein was detected by Western blot. In vivo experiment (1) 60 healthy Kunming male mice were randomly divided into 3 groups with 20 mice in each group. Chronic cadmium poisoning group (CD group): cadmium chloride solution 2mg/kg was subcutaneously injected twice a week, chronic cadmium intoxication plus vitamin E group (CD VE group) was injected with cadmium chloride, and vitamin E solution was given orally for 10 mg/kg daily. Control group: normal saline was injected. (2) after 13 weeks of modeling, the learning and memory ability, the activity of SOD and the content of MDA in the homogenate of cerebral cortex were measured by Y-shaped maze, and the changes of Nissl body of neurons in frontal cortex were observed by Nissl staining. The ultrastructural changes of neurons in frontal cortex were observed by transmission electron microscope, and the expression of caspase-3 protein in cerebral cortex was detected by immunohistochemistry, and the positive cells of GFAP and caspase-3 were detected by Western blot. Results (1) the purity of primary cortical neurons and astrocytes cultured in vitro was over 90%, (2) compared with the control group, the concentration of cadmium increased and the time of exposure to cadmium prolonged. The morphological damage of neurons and astrocytes was gradually aggravated, and the morphological damage of neurons and astrocytes was alleviated after the addition of vitamin E. The increase of SOD activity and the decrease of MDA content (P0.05) increased the expression of caspase-3 protein in Bcl-P / Bax ratio (P0.05) compared with the control group. There was still significant difference in the indexes (P0.05). The learning and memory ability of mice in the CD group was significantly lower than that in the control group and CD VE group (P0.05). The activity of); (in the cerebral cortex of the mice in the CD VE group was lower than that in the control group and CD VE group, and the content of); (in the CD VE group was higher than that in the control group and CD VE group. Compared with the control group and the CD VE group, the Nissl bodies of the frontal cortex neurons in the CD and C groups were decreased, while those in the VE group were significantly lower than those in the control group and CD VE group. The expression of caspase-3 protein in cerebral cortex was significantly higher than that in the control group (P0.05), and the neuron Nissl body in the CD VE group was slightly decreased compared with the control group. The expression of caspase-3 protein in cortex was also higher than that in control group (P0.05). Conclusion Vitamin E has a protective effect on the injury of cerebral cortex neurons and astrocytes induced by cadmium, and the protective effect of vitamin E is related to its antioxidation and the decrease of apoptosis.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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