【作者】 佟强；

【导师】 石静萍；

【作者基本信息】 南京医科大学 ， 神经病学， 2009， 硕士

【摘要】 AT1R拮抗剂对Alzheimer病动物学习记忆及tau蛋白磷酸化影响的研究阿尔茨海默病(Alzheimer disease, AD)是以进行性记忆损害、认知障碍和行为改变为主要表现的慢性神经退行性疾病,是老年痴呆中最常见的类型。随着人口预期寿命的延长,其在老年人口中的发病率和患病率逐年提高。由于缺乏确实有效病因治疗和预防的药物因而带来严重的经济、社会负担,从而成为世界各国神经科学和神经药理学研究热点。生物化学、生理学以及功能研究发现脑内存在独立而完善的局部肾素-血管紧张素系统(rennin-angiotensin system,RAS),血管紧张素Ⅱ(AngⅡ)为RAS内最重要的活性成份,主要通过与其1型受体(angiotensinⅡtype 1 receptor, AT1R)和2型受体(angiotensinⅡtype 2 receptor , AT2R)结合发挥作用,使用选择性AT1R和AT2R拮抗剂及靶基因研究证实RAS的绝大部分功能通过AT1R介导。Savaskan等人通过免疫组织化学的方法研究发现AD患者大脑顶叶皮质ACE、AngII和AT1R表达水平增加,此外还发现皮质血管周围的AngII和ACE活性增强,该研究表明AD患者脑内RAS在发病过程中被激活,在另外的一些研究中也同样得到证实。这为AngⅡ参与AD发病提供了分子病理基础。此外,AD动物模型研究发现持续RAS激活可致肾素/血管紧张素原转基因鼠认知功能损害,而AT1R拮抗剂可以改善AD模型鼠的学习与记忆功能,这些结果提示AngⅡ可能通过其1型AngⅡ受体(angiotensinⅡtype 1 receptor,AT1R)介导参与AD的发生和进展过程,是AD干预的潜在靶点。而动物实验研究发现AT1R拮抗剂改善胆碱能神经元功能障碍提高动物学习记忆能力,大量临床试验研究也发现AT1R拮抗剂延缓AD患者学习记忆损害降低AD的患病率;提示RAS参与AD发病,但具体的机制仍不清楚。因此本研究着重从胆碱能系统和tau蛋白磷酸化探讨AT1R拮抗剂在AD中的保护作用机制。本实验包括以下两个部分:第一部分:厄贝沙坦对Aβ1-42侧脑室注射大鼠学习记忆能力及海马tau蛋白磷酸化的影响目的研究厄贝沙坦对Alzheimer病(AD)大鼠学习记忆能力及海马tau蛋白磷酸化的影响。方法侧脑室注射寡聚体形式的Aβ1-42制作AD大鼠模型;厄贝沙坦干预组给予厄贝沙坦30mg·kg-1·d-1灌胃,AD模型组给予等量生理盐水灌胃,自术前一周至术后2周连续给药,手术当天暂停给药一次。用Morris水迷宫试验检测学习记忆能力,Western blot法检测海马tau蛋白丝氨酸199/202位点、396位点磷酸化;分析软件测定western blot图像条带光密度(OD)值,单因素方差分析比较各组间差异。结果(1) AD模型组Morris水迷宫试验逃避潜伏期比正常对照组和假手术组长(P<0.05),厄贝沙坦干预组与正常对照组、假手术组及模型组差异无统计学意义。(2)4组间海马tau蛋白总量差异无统计学意义;(3)与正常对照组和假手术组相比,AD模型组丝氨酸199/202(tau1)位点和396位点磷酸化tau蛋白含量明显增加(均P<0.05),厄贝沙坦干预组丝氨酸tau1和396位点磷酸化tau蛋白水平比AD模型组明显低(P<0.05)。结论厄贝沙坦能减轻AD大鼠海马tau蛋白磷酸化水平。第二部分:氯沙坦对Aizheimer病转基因小鼠学习记忆能力及胆碱能系统酶活性的影响目的研究氯沙坦干预对Aizheimer病转基因小鼠学习记忆能力及胆碱能系统酶活性的影响。方法5月龄雌性APPswePS1dE9转基因阳性小鼠随机分为阳性对照组、低剂量组、中剂量组、高剂量组,雌性APPswePS1dE9阴性纯合子为阴性对照组,氯沙坦溶于饮用水分别配成0.6g·L-1、0.2g·L-1、0.06 g·L-1,对照组给予正常饮用水,连续给药3个月;用Morris水迷宫试验检测学习记忆能力,分光光度法检测小鼠皮质乙酰胆碱转移酶(choline acetyltransferase, ChAT)和乙酰胆碱酯酶(True choline esterase,TChE)活性,单因素方差分析比较各组间差异。结果与其它各组相比高剂量组小鼠平均潜伏期明显缩短(P<0.01),而其它各组间无统计学差异。Losartan治疗组ChAT活性呈剂量依赖性增加,高剂量组ChAT活性显著性增加(与对照组比P<0.01,与低剂量组比P<0.05);与阴性对照组相比,低剂量组和中剂量组ChAT活性也明显上调(P<0.05);而两对照组间无统计学差异;各组间TChE活性无统计学差异结论氯沙坦提高转基因小鼠学习记忆能力和改善胆碱能神经元功能更多还原

【Abstract】 Alzheimer disease （AD） is an age-related chronic neurodegenerated disease featured by progressive memory impairments, cognitive disorder and behavioral change as the most commen type of senile dementia. As the prolongation of life expectancy, its incidence and morbidity rate in aging population keeps increasing yearly. Due to lacking of effective and definite medicine in etilogical treatment and prevention, it spells serious economical and social burden and thus becomes hot spot of neuroscience and neuropharmacology.Biochemical, physiological and functional studies suggest existence of an independent and well-developed focal RAS, in which angiotensinⅡ（AngⅡ） as the most important effector of RAS,produce a marked effect majorly by binding to angiotensinⅡtype 1 receptor （AT1R） and angiotensinⅡtype 2 receptor （AT2R）. Selective angiotensinⅡreceptor blockage and target gene researches suggest that RAS implements most function majorly by AT1R.Savaskan et al found AngⅡ、angiotensin convert enzyme（ACE） and AT1R elevated in parietal cortex of AD patient by immunohistochemical method, as wellas strengthened activity of perivascular AngⅡand ACE in cortex. These suggested RAS in the brain was activated during onset of AD and the result was also confirmed in several other experimental studies. These have provided the molecule pathological mechanism basis for Ang II participating in AD morbidity. But besides, the AD animal model studies find that sustained RAS-activating cause the rennin-angiotensinogen transgenic mouse cognition function damage, AT1R blockage can be able to improve study ability and memory function of AD model mouse, these results point out that Ang II maybe participates in AD’s happening and the process progressing, and will be the potent target spot that drug interferes with AD. At the same time animal experimental researches discovered AT1R blockage could improve the cholinergic neuron dysfunction and learning memory ability; generous clinical trials also found AT1R blockage could delay memory impairment in AD patient and decrease the morbidity rate of AD. It is evident that RAS participate in onset and development of AD, but the precise mechanism remains unknown. So that this study focused on the protection mechamism of AT1R blockage on cholinergic neuron system and tau phosphorization in AD animals. This stude includes the following two parts. PartⅠInfluence of Irbsartan on memory ability and tau protein phosphorylation on the hippocampus of rats injected Aβ1-42 into cerebral ventrilesObject:To explore the effect of Irbesartan on memory ability and tau protein phosphorylation in the hippocampus of Alzheimer disease rats.Methods:AD rat models were established by injection aggregatedβ-amyloid （1-42） into the cerebral ventricle. Irbesartan at dose of 30mg·kg^-1·d^-1 was administered to Irbesartan-intervention group by intragastric administration .AD model group was given equal physiologic saline by intragastric administration. The administration lasted from 1 week before surgery to 2 weeks after surgery, suspended at the day of surgery. Morris water maze trail was performed to test learning and memory ability. Western blotting was carried out to observe the tau protein phosphorylation. Analytic software was used to determine the OD value of protein strap. Parameters ware analyzed for intergroup differences by one-way ANOVA.Results: The escape latency of AD model group was prolonged （P<0.05） compared to normal control group and sham operation group, while the escape latency of intervention group was not different statistically from normal control group and sham operation group . No difference in total tau protein was observed in four groups ;compared with normal control group and sham operation group, the Ser^199/202Tau protein and the Ser³⁹⁶Tau protein in AD model group were significant increased（P<0.05）. Compare with model group, the Ser^199/202Tau protein and the Ser³⁹⁶Tau protein in intervention group were obviously decreased（P<0.05）;But no statistical difference was observed between intervention group and the other 2 groups. Conclusion:Irbesartan can attenuate the level of tau protein phosphorylation in the hippocampus of Alzheimer disease rats PartⅡEffect of Lostarn on memory ability and cholinergic neuron system enzyme action in Aizheimer disease transgenic miceObject:To investigate the effect of Losartan on memory ability and cholinergic neuron system enzyme in Alzheimer disease transgenic mice.Methods: Fiive–month-old female transgenic mice were randomly divided into positive control group、low-dosage group、middle-dosage group and high-dosage group. Age-matched female negative homozygote mice were taken as negative control group. Losartan at a different dosage of 0.6g·L-1、0.2g·L-1 and 0.06g/L was dissolved into drinking water and administered to different dosage groups for three months;Two control groups received normal drinking water. Morris water maze trail was performed to test learning and memory ability. Spectrophotometer was carried out to observe the activity of acetyltransferase （ChAT） and True choline esterase （TChE）. Parameters ware analyzed for intergroup differences by one-way ANOVA. Results: The escape latency of high-dosage group was significantly shortened （P<0.01） compared to the other groups ; While no significant difference was observed among the other groups.The activitity of ChAT in losartan therapy group showed a dosage dependent elevation , and that in high-dosage group was found significantly increased compared to that in control groups（P<0.01） and the low-dosage group（P<0.05）, although the activity of the low-dage group and the middle-group were upregulated （P<0.05）compared with the negative control group, there was no statistical difference between the negative group and the positive group. No statistical difference on TChE activity was observed among 5 groups.Conclusion:Losartan may improve the learning and memery ability and cholinergic neurons function in transgenic mice.更多还原