【作者】 房恒通；

【导师】 蒋与刚；

【作者基本信息】 中国人民解放军军事医学科学院 ， 营养与食品卫生学， 2007， 硕士

【摘要】 目的：建立认知与运动功能损伤大鼠模型；观察复合营养素配方对认知与运动功能损伤大鼠的改善效果，并初步阐明其作用机制。方法：(1)海马内注射β淀粉样蛋白(Aβ)致认知与运动功能损伤大鼠模型的探讨。3～4月龄雄性SD大鼠27只，随机分为正常对照组、Aβ组和生理盐水组，每组9只。正常对照组不作任何处理，Aβ组每侧海马内注射1μl Aβ，生理盐水组海马内注射等体积生理盐水。海马内注射两周后，观察海马病理学改变；水迷宫、避暗实验检测大鼠的学习、记忆功能；荧光分光光度法测定血清维生素A、E含量；紫外分光光度法测定血清维生素C含量；化学发光竞争免疫法测定血清叶酸含量；原子吸收分光光度法测定血清铁、铜、锌含量；气相色谱法测定血清游离脂肪酸含量；全血谷胱甘肽还原酶、红细胞天门冬氨酸转氨酶活性、血清和脑组织乙酰胆碱酯酶(AChE)活性也采用紫外分光光度法。(2)老龄大鼠作为认知与运动功能损伤动物模型的研究。雄性Wistar大鼠16只，分为老龄组(15月龄)和青年组(3月龄)，每组8只。水迷宫、rod walking及plank walking实验检测大鼠的行为学功能。(3)复合营养素对老龄大鼠认知与运动功能的干预作用及机制研究。选取15月龄Wistar大鼠，雄性14只，雌性16只，随机分为干预组和对照组，每组雄性7只，雌性8只。干预组大鼠饲喂添加复合营养素的自制饲料，同时灌胃植物提取物混合液，而对照组饲喂基础饲料，同时灌胃蒸馏水。灌胃剂量为4ml／(kg bw·d)。实验干预期为10周。采用水迷宫、rod walking实验检测大鼠的认知及运动行为；荧光分光光度法检测脑组织去甲肾上腺素(NE)、5-羟色胺(5-HT)、多巴胺(DA)、血清VE含量；紫外分光光度法测定脑组织乙酰胆碱(ACh)含量，血清总蛋白含量、白蛋白含量，脑组织及血清AChE活性，全血谷胱甘肽过氧化物酶(GSH-Px)活性，血清总抗氧化能力(T-AOC)、抑制羟自由基能力；神经电生理法观察海马齿状回长时程增强效应的改变；二维凝胶电泳(2-DE)检测大鼠额叶皮层蛋白质表达的变化，利用肽质量指纹图谱分析和数据库检索鉴定表达差异的蛋白，Western blotting验证stathmin 1蛋白的表达变化。结果：(1)未观察到Aβ组海马组织Aβ沉着及明显的病理变化。与正常对照组比较，Aβ组学习、记忆功能有下降趋势，但差异无显著性；Aβ组血清VC、铁含量显著下降；血清及脑组织AChE活性显著升高；血清游离脂肪酸构成发生显著改变。(2)与青年组大鼠比较，老龄组大鼠认知与运动功能下降。(3)与对照组比较，干预组老龄大鼠发生以下改变：①认知与运动功能得到改善；抗氧化能力增强，血清VE含量增加；脑组织ACh、NE、5-HT、DA含量升高，而血清及脑组织AChE活性显著下降；②海马齿状回长时程增强幅度明显提高，海马突触传递效能增强；③额叶皮层2-DE图谱中11个蛋白点含量发生变化。质谱鉴定的10种蛋白质中，其中6种蛋白质含量降低，分别为：丙酮酸激酶、丙酮酸脱氢酶E1α1、天门冬氨酸转氨酶、线粒体肌酸激酶1、电压依赖性阴离子通道2、磷酸丙糖异构酶1；4种蛋白质含量升高，分别为：过氧化物氧化还原酶2、线粒体H-ATP合酶d亚单位、stathmin 1、Cu-Zn超氧化物歧化酶。④额叶皮层stathmin 1蛋白的表达量升高。结论：(1)老龄大鼠可作为认知与运动功能损伤的动物模型研究复合营养素的干预效果及其机制。(2)复合营养素对老龄大鼠的认知与运动功能有改善作用，其作用途径可能与改善大鼠的抗氧化状态，增加与行为功能密切相关的神经递质含量，改善神经突触之间的传递效率，上调与认知相关蛋白质的表达等有关。其中，stathmin1蛋白可能对老龄大鼠神经细胞有一定的保护作用。更多还原

【Abstract】 Objective: To investigate the improved effects of combined nutrients in rats with impaired cognition and motor function and illustrate its possible mechanism.Method: (1) The model with impaired cognition and motor function was established by intrahippocampal injection ofβ-amyloid protein (Aβ). Twenty seven male Sprague-Dawley rats were randomly divided into control group,βamyloid group (Aβ) and saline group. The rats were injected with Aβor saline in bilateral hippocampus (1μl per lateral) in Aβand saline group respectively. Two weeks later, the pathologic changes of hippocampus were observed. The learning and memory ability of rats were examined by water maze test and one trial passive avoidance test. The contents of serum vitamin A and E were determined by fluorospectrophotometry, vitamin C by ultraviolet spectrophotometry, folic acid by chemiluminescence competitive immunoassay, iron, copper and zinc by atomic absorption spectrophotometry and free fatty acids by gas chromatography. The activity of glutathione reductase in blood, aspartic transaminase in erythrocyte and acetylcholine esterase in serum and brain was also determined by ultraviolet spectrometry. (2) Sixteen male Wistar rats were divided into aged group (15months old) and young group (3months old), eight in each. The behavioral tests were carried out through water maze, rod walking and plank walking test. (3) Fourteen male and sixteen female Wistar aged rats were randomly divided into intervened group and control group, seven male and eight female in each. The rats in intervened group were fed with diets supplemented with combined nutrients and mixed plant extract administered intragastrically at 4ml/kg bw/d, while the rats in control group were fed with basic diets and water. The experiment lasted 10 weeks. The cognition and motor behavior was tested with water maze and rod walking test. The levels of noradrenaline, serotonin, dopamine in brain and vitamin E in serum were determined by fluorospectrophotometry, brain acetylcholine, serum protein and albumin, activities of brain and serum acetylcholine esterase, blood glutathione peroxidase, serum total anti-oxidation capability and hydroxy radical inhibition capability by ultraviolet spectrophotometry. The changes of long-term potentiation in hippocampal dentate gyrus were observed through electrophysiology, and protein synthesis in frontal cortex by two dimensional gel electrophoresis. The expressed differential proteins were identified by analysis of peptide mass fingerprint and database retrieval. Western blotting was used to identify the changes of stathmin 1 expression. Results: (1) Deposition of Aβand pathological changes were not observed in hippocampus in Aβgroup. Compared with control group, the learning and memory in Aβgroup decreased but it’s insignificant. The contents of serum vitamin C and iron decreased in Aβgroup, while the activity of acetylcholine esterase in brain and serum increased significantly.The composition of free fatty acids in serum changed significantly. (2)Compared with young rats, the cognition and motor function of aged rats declined, but restored after intervention by combined nutrients. (3) In intervened group, the anti-oxidation capability and vitamin E contents in serum, the contents of acetylcholine, noradrenaline, serotonin and dopamine in brain were increased, but acetylcholine esterase activity in serum and brain decreased. The amplitude of long-term potentiation in hippocampal dentate gyrus and the efficacy of hippocampal synaptic transmission were significantly elevated. Eleven protein spots with changes of contents were observed in two dimensional gel electrophoresis maps and ten of them were identified successfully. Among the identified proteins, six proteins decreased in frontal cortex of dietary supplemented rats including pyruvate kinase, pyruvate dehydrogenase E1 alpha 1, aspartate transaminase, mitochondrial creatine kinase 1, voltage-dependent anion channel 2 protein and triosephosphate isomerase 1, while the other four proteins increased, including peroxiredoxin 2, subunitd of mitochondrial H-ATP synthase, stathmin 1 and Cu-Zn superoxide dismutase. The results of Western blotting confirmed that frontal cortex stathmin 1 in intervened group increased than that in control group. Conclusion: The aged rat can be taken as model with impaired cognition and motor function to study the effects of nutrition intervention and the mechanisms involved. The combined nutrients have improved effects on cognition and motor function of aged rats and the effects might correlate with improved anti-oxidation status, elevated neurotransmitter level, increased efficacy of synaptic transmission and increased expression of protein related to cognition in frontal cortex. Stathmin 1 might protect the neuron in aged rat.更多还原