【作者】 方传勤；

【导师】 周华东；

【作者基本信息】 第三军医大学 ， 神经病学， 2007， 博士

【摘要】 阿尔茨海默病(Alzheimer’s disease)是引起老年人痴呆的主要原因,已经成为危害老年人群健康和生命的重要疾病。近年研究表明血管危险因素在AD发生、发展中起着重要作用,干预和减少血管危险因素对于预防和延迟AD的发生具有重要意义。在血管危险因素中,中年时期的高胆固醇血症与老年时期的AD关系最密切。研究证实高血清胆固醇的个体具有更高的AD易感性,在高脂肪和高热量饮食国家AD的发病率高,低脂肪饮食国家AD的发病率低。有研究发现AD病人的血浆总胆固醇和LDL胆固醇水平升高,HDL水平下降。与对照组相比,服用他汀类药物的人AD发病率降低70 %,为胆固醇代谢异常导致AD提供了一个非常有力的证据。在体实验报道高胆固醇促进β淀粉样蛋白前体的表达、淀粉样蛋白生成,和增加β淀粉样蛋白的沉积,而改用普通饮食可以减少Aβ的生成。高胆固醇破坏突触结构功能的可塑性,改为普通饮食后突触的损伤得以逆转。那么,高胆固醇血症对AD患者的认知功能影响如何?这一方面研究甚少。中枢神经系统炎性反应是AD早期病理表现,AD病人在严重认知功能下降之前,已经出现老年斑激活的小胶质细胞和星形胶质细胞,激活的小胶质细胞和星形胶质细胞可释放各种细胞毒性分子,包括活性氧、一氧化氮、促炎症细胞因子、蛋白水解酶、补体和兴奋性氨基酸。实验显示胆固醇喂养的兔子脑中出现炎性反应和AD样病理改变,高胆固醇诱导小胶质细胞和星形胶质细胞活化,研究发现高胆固醇可增加APOE基因敲除鼠和野生鼠脑中主要炎症介质IL-6的表达,并且这一作用与APOE敲除与否无关。高胆固醇是否会加重Aβ诱导的炎性损伤呢?这一方面尚未见研究。神经原纤维缠结是AD最重要的病理特征之一。Tau蛋白是一种多功能的微管相关蛋白,能稳定微管,促进微管装配。异常过磷酸化的Tau蛋白构成神经原纤维缠结的核心。研究提示Aβ是AD病理过程的启动环节,Tau蛋白功能异常可能是神经元功能障碍和死亡的重要环节。目前对高胆固醇与Tau蛋白磷酸化关系研究较少,高胆固醇是否影响Aβ诱导的Tau蛋白异常磷酸化尚未见报道。对以上问题的研究有助于了解胆固醇影响AD发生和发展的机制,同时为通过控制胆固醇含量预防AD的发生和延缓AD的进展提供实验依据。因此,本研究的主要内容包括如下,探讨高胆固醇对AD大鼠认知功能的影响;高胆固醇对AD大鼠海马神经细胞损伤的影响;从炎性因子、Tau异常磷酸化角度探讨高胆固醇对AD大鼠脑海马区神经损伤的影响的机制。方法:1高胆固醇对AD大鼠认知功能影响及其机制SD大鼠(雄性,体重250±20g)随机分成四组:高胆固醇饮食+AD组,AD组,高胆固醇饮食+Sal组,Sal组。高胆固醇饮食+AD组和高胆固醇饮食+Sal组喂养高胆固醇饮食,AD组和Sal组喂养标准饮食(普通鼠粮),分别喂养8周。将凝聚态Aβ1-40注射入高胆固醇饮食+AD组和AD组大鼠右侧海马齿状回建立AD大鼠模型,将生理盐水注入高胆固醇饮食+Sal组和Sal组大鼠右侧海马齿状回。采用Morris水迷宫和穿梭箱检测大鼠学习记忆;使用HE染色观察病理改变;尼氏染色检测神经元缺失;刚果红染色观察Aβ的沉积。利用免疫组织化学方法检测GFAP、IL-6、TNF-α和Tau蛋白水平;使用原位杂交和RT-PCR检测IL-6和TNF-αmRNA水平。2高胆固醇对Aβ1-40诱导神经元损伤和胶质细胞活化影响的体外研究用2d龄SD大鼠海马建立神经组织混合培养体系。分为3组:正常对照组、Aβ(2uM)组、胆固醇(1mM)+ Aβ(2uM)组。检测细胞培养上清液LDH释放度,IL-6、TNF-α的含量、免疫荧光染色检测神经元形态和数量变化。结果:1高胆固醇对AD大鼠认知功能影响及其机制的在体研究.高胆固醇饮食AD组在Morris水迷宫和穿梭箱都表现出严重的学习和记忆损害。在莫里斯水迷宫实验中,AD以及其他各组相比,高胆固醇饮食+AD组具有较长的平均逃避潜伏期和较少的穿环数和较小的平台象限游泳距离百分比。在穿梭箱实验中,与AD组以及其余各组相比,高胆固醇饮食+AD组有较少主动回避率和总回避率,较长回避潜伏期。在各组中,高胆固醇饮食+AD组神经元缺失最大,神经元缺失显著多于标准饮食AD组。在相同饮食情况下,AD组神经元缺失较Sal组多。在相同注射试剂情况下,高胆固醇饮食组神经元缺失重于标准饮食组。与其余各组相比,高胆固醇饮食+AD组在海马齿状回和CA1区,有更多的IL-6和TNF-α阳性细胞,与AD组相比,差异显著。皮质部位IL-6和TNF-α阳性细胞只与饮食有关,与注射试剂无关,高胆固醇饮食组多于标准饮食组。RT-PCR结果显示高胆固醇饮食+AD组IL-6mRNA和TNF-αmRNA多于标准饮食AD组。高胆固醇饮食诱导海马齿状回和CA1区(Pser202)Tau表达,高胆固醇饮食+AD组(Pser202)Tau阳性细胞数多于AD组。高胆固醇饮食+AD组(Pser202)Tau阳性细胞数最多。2高胆固醇对Aβ1-40诱导神经元损伤和胶质细胞活化的影响Aβ组细胞培养上清液中LDH释放度较正常对照组明显增高,高胆固醇+Aβ组LDH释放度在各组中最高,与Aβ组相比,差异显著。神经元荧光染色结果显示,正常对照组神经元胞体饱满,神经突起较长,神经突起形成网络联系;Aβ组与正常对照组相比,神经元数目减少,突起变短;高胆固醇+Aβ组神经元数目减少最明显,神经突起断裂,突起回缩。正常对照组细胞培养上清液中有少量IL-6和TNF-α分泌,Aβ组IL-6和TNF-α含量明显增高,在各组中高胆固醇+Aβ组含量最高,与Aβ组相比有非常显著差异。结论:1.高胆固醇加重阿尔茨海默病大鼠认知功能损害。2.高胆固醇增加AD大鼠海马IL-6和TNF-α的表达和Tau异常磷酸化,导致神经元缺失增加,进而加重认知功能损害。3.高胆固醇促进Aβ诱导体外培养的神经元损伤和IL-6和TNF-α分泌。体外研究证实高胆固醇增强Aβ的神经元损伤和胶质细胞活化作用。更多还原

【Abstract】 Alzheimer’s disease is the most common form of dementia in later life, and a major cause of disability and death in the elderly. Recently study indicates vascular risk factors play important role in the pathogenesis and development of Alzheimer’s disease. It is important to interfere in and reduce modifiable vascular risk factors to prevent and delay Alzheimer’s disease.In all vascular risk factor, hypercholesteremia in midlife have the closest relation with AD in older age. There have been accumulating evidences pointing toward a potentially important link between high cholesterol, Aβ, and AD. Evidences from clinical and epidemiological studies suggest that high cholesterol might influence the development of AD. There are several investigations demonstrating that individuals with elevated serum cholesterol levels have increased susceptibility to AD. The prevalence of AD is higher in countries with high-fat and high-calorie diets and lower in those with low-in-fat diets. In addition, recent other studies have indicated that the prevalence of AD is reduced among people taking a class of cholesterol-lowering medicines. Experimental studies provided additional and important support for the role of cholesterol in AD. Several studies in vitro have reported that modifications of cholesterol content can influence the expression of the amyloid precursor protein (APP) and alter Aβproduction, and there had also circumstantial evidence that high cholesterol could change Aβdeposition. Cholesterol-enriched diet can destroy synaptic plasticity, but regular diet can reverse the impaiment of synaptic plasticity. Then, whether high cholesterol further aggravate AD patient’s pathogenetic changes, and how to influence their cognition function are not fully understood.Inflammatory reaction in the central nervous system is an early pathological change in the brains of AD patients. In addition, there have been clear evidences of inflammatory process in the AD brain. Neuritic plaques activate microgial cell and astrocytes even before severe cognitive decline. A lot of active microgial cells and reactive astrocytes have the potential roles to injure neurons by the release of different cytotoxic factors including reactive oxygen species, nitric oxide, proinflammatory cytokines, proteolytic enzymes, complement factors, and excitatory amino acids et al. Evidences suggested that cholesterol-fed induced development of inflammation and AD-like pathological changes in rabbit brain. Another study shows high cholesterol induces the activation of both microglia and astrocyte in mice. High cholesterol induced expression increase of IL-6. Whether high cholesterol can increase overall inflammatory damage induced by Aβis unclear.Neurofibrillary tangles (NFTs) is a common feature of Alzheimer’s disease (AD). Tau is a multifunctional microtubule-associated-protein, which can stable microtubule and promote assembly of microtubule. Hyperphosphorylated Tau is major component of neurofibillary tangles. Studies show Aβis trigger of pathogenesis and abnormal function of Tau, which results in functional disturbance and death of neuron. There are few studies of relation between high cholesterol and Tau hyperphosphorylation, effect of high cholesterol on Tau hyperphosphorylation induced by Aβhas not been reported.It is necessary to understand the mechanism of influence of high cholesterol on the pathogenesis and development of AD, and to supply experimental base for preventing and delaying AD by change of cholesterol content. Thus in the present study, there are main contents including effects of high cholesterol diet on cognitive impairment by Aβinjection in hippocampus of rats; By studies in vive and vitro, evaluate the effects of high cholesterol on neurons loss in hippocampus of AD rats; influences of high cholesterol on expression of IL-6, TNF-αand phosporylation of Tau induced by Aβ.Method:1. Effect of high cholesterol on Aβ-induced cognitive impairment and its’mechanism. SD rats (male, weight 250±20g,) were divided into four groups randomly: CH+ AD group,AD group, CH+Sal group, Sal group. Each group has eight rats. CH+AD group and CH+Sal group rats had eaten high cholesterol diet, AD group and Sal group rats had Standard rat diet for eight weeks, then aggregated beta-amyloid1-40 was injected into right hippocampus of CH+AD group and AD group to establish AD rat modles, Sal was injected into right hippocampus of CH+Sal and sal group rats. Morris water maze and shuttle box test were measured rat learning and memory function; The pathological changes were observed with HE staining; Nissle’s staining was used to assessed loss of neuron; Aβ depositon was evaluated by congo red staining; GFAP, IL-6, TNF-αand (Pser202)Tau protein expression were analyzed by immunohistochemistry; IL-6 and TNF-αmRNA were further detected by in suit hybridization and RT-PCR.2. Effect of high cholesterol on Aβ-induced injury of neurons and activation of glial cell in vitro.The mixed hippocampal cells of 2d-old Sprague-Dawley rats were cultured. There were three groups: normal control group, Aβ(2μM) group, cholesterol (1mM)+Aβ(2μM) group. Lactic dehydrogenase (LDH) releasing assay was applied for measurement of the change of neuron cell. IL-6 and TNF-αcontents in culture supernatants were determined by ELISA and neurons were subjected to fluorescence staining.Result:1. Effect of high cholesterol on Aβ-induced cognitive impairment and its mechanism. CH+AD rats exhibited seriously learning and memory deficits in both Morris water maze and shuttle box. In Morris water maze test, mean value of escape latency of CH+Aβgroup was longer than the other groups, CH+AD group had shorter distance percentage of spending in the former platform quadrant and fewer frequency of crossing the former platform site than the SD+ Aβand other groups. In shuttle box, CH+AD group had fewer AAR, GAR and longer escape latency, comparing with AD and other group. It suggested high cholesterol aggravated cognitive impairment of AD rats.CH+AD group rats showed most significant defects of neurons in all groups. With the same food, Aβ-injected rats had greater neuronal loss than Sal rats. With the same injection, high cholesterol fed rat had more neurons loss than standard diet.IL-6 and TNF-αpositive cells of dentate guys and CA1 were more in CH+AD group rats than the other groups; There were more IL-6 and TNF-αpositive cell in cortex of high cholesterol diet group than standard diet group. In RT-PCR trail, expression of IL-6 mRNA and TNF-αmRNA is higher in CH+AD group rats than AD groups.High cholesterol induced increase of (Pser202)Tau positive cells in dentate guys and CA1, CH+AD rats had the most number of (Pser202)Tau positive cell.CH+AD rats had significantly more (Pser202)Tau positive cell than AD rats.2. Effect of high cholesterol on Aβ-induced injury of neurons and activation of glial cell in vitro. Percent of LDH release in hippocampal culture of Aβgroup is larger the control group. In all groups, CH+Aβhas the largest percent of LDH release, and larger than Aβgroup.In the normal control group, fluorescence stains of the neuron were even, neuronal body was full, nerve processes are long and formed a network. In the Aβgroup, the number of neurons decrease and nerve processes shorten, CH+Aβgroup has fewer neurons and shorter nerve process than Aβgroup.IL-6 and TNF-αcontents in culture supernatants of normal control group are low. IL-6 and TNF-αcontents of Aβgroup significantly increase, in all group, IL-6 and TNF-αcontents of CH+Aβgroup is highest, the differences in IL-6 and TNF-αcontents were significant between CH+Aβgroup and Aβgroup.Conclusion:1. High cholesterol may deteriorate cognitive impairment caused by Aβinjection into hippocampus of rats.2. High cholesterol increases the expression of tow important inflammatory cytokines IL-6 and TNF-α,and phosphorylation of (ser202)Tau in hippocampus of AD group,which causes more neurons defect and severer cognitive impairment.3. high cholesterol facilitates Aβ-induced injury of cultured neurons and activation of glial cells, which secrete IL-6 and TNF-α.Experiment in vitro further confirms that high cholesterol enhance injury of neurons and activation of glial cells induced by Aβ.更多还原