【作者】 王威；

【导师】 唐一源；

【作者基本信息】 大连理工大学 ， 神经信息学， 2009， 博士

【摘要】 氧化应激和心理应激是人类正常生命进程中不可避免、与生理病理密切相关的事件,二者与老年性痴呆病理特征β-淀粉样蛋白(β-amyloid protein,Aβ)的生成密切相关。然而对于氧化应激、心理应激尤其是二者共同作用时脑内Aβ1-42及其代谢途径中相关分子的变化,目前尚未完全阐明。为了探讨这个问题,我们进行了如下研究工作:首先,确定D-半乳糖致氧化应激损伤的适宜剂量。根据文献统计结果,选择75、120、150和1000mg/kg剂量的D-半乳糖,连续8周注射,观察小鼠认知功能及脑内氧化酶学指标变化。结果表明,四个剂量D-半乳糖均能导致小鼠认知功能损伤,脑内超氧化物歧化酶(superoxide dismutase,SOD)、过氧化氢酶(catalase,CAT)活性下降和丙二醛(malondialdehyde,MDA)含量增加,但对不同指标损伤程度不同,结合小鼠给药期间的一般状态变化,我们选取120mg/kg做为氧化应激模型适宜剂量。其次,通过D-半乳糖建立氧化应激模型,急性束缚建立心理应激模型,采用行为学、生化检测、放射性免疫和免疫组化等方法,探讨氧化应激、心理应激尤其是二者共同作用对小鼠认知功能和脑内Aβ1-42表达的影响。三个应激组小鼠脑内SOD和CAT活性降低,MDA增加,心理应激和复合应激组小鼠血清皮质酮含量升高,表明氧化应激和心理应激模型是可行性的。三个应激组小鼠出现不同程度认知功能损伤,其中氧化应激对空间认知损伤明显,心理应激和复合应激对空间和非空间认知都有损伤。三个应激组中小鼠脑内Aβ1-42表达均有增加,而且复合应激组较单一应激组增加更明显。最后,探讨脑内Aβ1-42表达增加可能的代谢机制。采用western blotting和RT-PCR方法,考察脑内Aβ1-42的生成、降解和转运途径中主要信号分子的改变。结果表明,三种代谢途径都参与了不同应激条件下脑内Aβ1-42的代谢,其中BACE1表达上调可能是氧化应激和心理应激诱导脑内Aβ1-42生成增加的主要途径;MMP-2和IDE表达的增加,说明降解过程参与了应激条件下脑内Aβ1-42的清除;氧化应激和心理应激组中IDE表达不同,提示在不同应激原作用下,发挥作用的降解酶及作用程度可能不同;三个应激组中LRP-1表达增加,RAGE表达变化不明显,说明氧化和心理应激下,脑内Aβ1-42会向脑外转运,而脑外Aβ不会向脑内转运,可能是机体在应激刺激下的保护机制。复合应激能增加单一应激中BACE1的表达,可能与氧化应激和心理应激共同作用有关。复合应激中MMP-2和LRP-1表达较单一应激明显增加,有利于机体清除Aβ1-42。更多还原

【Abstract】 Oxidative and psychological stresses are unavoidable pathological and physiological events correlated withβ-amyloid(Aβ) which is one of pathological characters of AD.It is still unknown whether oxidative or(and) psychological stressors might influence Aβ1-42 expression and its metabolism-associated molecules in brain.To elucidate these questions, bellowed studies were performed:First,to determine a suitable dosage of D-galactose(D-gal) for oxidative stress. According to the statistical data of references,we chose dosages of 75,120,150 and 1000mg/kg for 8-week injection and explore the effects of D-galactose on anti-oxidative enzymes and cognitive functions.The results suggested that D-galactose of four dosages could induce significantly cognitive defects,decrease of superoxide dismutase(SOD) and catalase(CAT) activities,increase of malondialdehyde(MDA) content.However.the effect of D-galactose on indicators was different in each dosage.We finally chose 120mg/kg as the most suitable dosage according to mentioned above detections combining with general conditions of the mice during D-galactose treatment.Second,acute restraint stress was used to establish psychological stress animal models and then to explore the effect of oxidative,psychological and combined stresses on cognition, anti-oxidative enzymes,corticosterone content and Aβ1-42 expression by the methods of behavior tests,biochemistry,radio-immunity assay and immunohistochemistry.The results indicated that the activities of SOD and CAT decreased,but MDA content increased in three stress groups.Moreover,psychological and combined stresses could enhance corticosterone levels.Therefore,the animal models of oxidative or(and) psychological stresses were feasible. D-galactose mainly impaired spatial cognition but acute restraint stress and combined stress could lead to both spatial and non-spatial cognition impairment.All the three stresses could upregulate Aβ1-42 expression and the increase in combined group was higher than any single stress group.Third.the study discussed the molecular mechanism of Aβ1-42 metabolism induced by D-galactose,acute restraint stress and combined stress by the methods of western blotting and RT-PCR.The results suggested that Aβ1-42 metabolism was involved in generation, degradation and transportation in which the increase of Aβ1-42 level mostly caused by upregulation of BACE1 expression.Matrix metalloproteinases-2(MMP-2) and insulin-degrading enzyme(IDE) were participated in degrading Aβ1-42.However,IDE increase in acute restraint stress but not D-galactose indicated that Aβ-degrading enzymes may be selectively role in different stress conditions.LRP-1 mediates a continuous efflux of brain Aβ1-42 into the circulation.This study found that LRP-1 in mice treated with D-galactose,acute restraint stress and in combination was higher than that of control. Furthermore,this study did not find any significant difference of RAGE expression between any treated group and control.The increase of LRP-1 and no change in RAGE may contribute to Aβ1-42 efflux and prevent Aβ1-42 accumulation in the brain.The higher expression of BACE1 in combination group than any of single stress group was perhaps associated with collaboration of D-galactose and acute restraint stress.Moreover,the higher expressions of MMP-2 and LRP-1 in combined stress group than that of in D-galactose or acute restraint stress group were compensatory for Aβ1-42 clearance.更多还原