【作者】 耿义群；

【导师】 徐小虎； 傅玉才；

【作者基本信息】 汕头大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 研究背景和目的衰老（Senescence）是随着年龄增长，机体各器官发生生物学功能退化的过程，表现为老年相关疾病的发病率增高，细胞更新的能力下降，死亡的可能性增加。热量限制（Calorie Restriction，CR）是一种目前公认的有效延缓衰老的方法。CR不仅能延缓衰老，还能延缓和预防一些与年龄相关疾病的发生发展。衰老的大脑在功能上主要表现为学习记忆能力减退，而CR能够减少与认知能力相关的疾病（如Alzheimer’s病）的发病率，但是CR对自然衰老引起的学习记忆能力衰减的延缓作用仍缺乏行为学和组织学依据，并且多数的研究结果是基于对动物进行终生饮食限制获得的，研究周期较长。对于CR是否能够延缓老年早期的大鼠学习记忆能力的衰退，还存有争议，甚至有人对成年或老年早期的大鼠进行饮食限制是否能够提高大鼠的生存率提出质疑。在明确CR能否对认知能力的下降有延缓作用时，确定认知相关的脑区（如海马和额叶皮质）神经元的衰老状态是揭示CR延缓学习记忆能力下降的细胞学基础，衰老细胞的鉴定是确定细胞是否发生衰老的标志。尽管有报道，在培养的细胞，衰老细胞表现为细胞变大，变扁平，但在组织学，衰老细胞仅从形态学上难以与其它细胞区分。生物学标记为衰老细胞的鉴定提供了一个重要方法，衰老相关的β半乳糖苷酶（Senescence associatedβ-galactosidase，SA-β-GAL）是目前被广泛采用的一种生物学标记物，它在衰老细胞中表达升高，因此成为鉴定细胞衰老的一个标志。值得提出的是，有些衰老的生物学标记并不具有普遍性，SA-β-GAL作为一个衰老的生物学标记在体外检测复制衰老的细胞已经得到了广泛的认可，但其是否能够应用于脑组织，作为检测衰老神经元的生物学标记，目前还未得到证实，因此在应用上应加以验证。本研究选定老年早期的大鼠，对其进行饮食控制，即选在认知能力衰退的年龄段进行观察，旨在明确限制饮食对老年早期的大鼠的生存状态的影响和通过行为学检测观察学习记忆能力的改变，利用SA-β-GAL在学习记忆产生的主要部位海马检测衰老神经元。探讨认知能力的提高是否与限制饮食引起的延缓神经元衰老有关，同时验证SA-β-GAL作为衰老的生物标记在脑组织检测衰老神经元的可靠性，为进一步探讨CR延缓海马衰老的生物学机制提供行为学和组织学依据。材料与方法实验动物选用18月龄的健康雄性Sprague Dawley大鼠，根据进食情况分为两组，一组为CR组（n=29）给予对照组进食量的60％的食量喂养，对照组为ad libitum（AL）组（n=31）自由进食，观察6个月，在此期间，记录大鼠的生存状况，每周测定大鼠体重；6个月后，进行开放场实验（open-field test）观察自发活动能力；利用Morris水迷宫（Morris water maze，MWM）检测大鼠的空间学习能力和参考记忆能力。选用6月龄（n=10），18月龄（n=6）和24月龄（n=7）的SD雄性大鼠，进行SA-β-GAL的组织化学检测；同时海马神经元原代培养，取培养6天，12天和20天的神经元进行SA-β-GAL的细胞化学检测。结果CR组给予60％的饮食控制后1-10周内体重增长率下降明显，低于对照组的近30％，之后CR组的体重增长率有缓慢的升高，并维持在低于对照组约20％左右（p＜0.001）。CR组的生存率明显高于AL组（p=0.039）；在开放场实验中测定的大鼠白发活动路程明显长于AL组（p=0.021），而自发活动速度也明显高于AL组（p=0.021），并且CR组大鼠具有在中央区域的活动距离长（p=0.048）和在周边区域活动速度快（p=0.012）的特点。Morris水迷宫定向航行实验中，CR组的逃避潜伏期（Escape latency，EL）明显比AL组短（p=0.038），而在空间探索实验中，比较两组的穿环次数无统计学差异（p=0.232），两组大鼠在目的象限Ⅲ中的时间和路程差别无统计学意义（p=0.324和p=0.367），但在象限Ⅱ中的时间和路程都有明显统计学差异（p=0.027和p=0.029）。与AL大鼠相比，CR组大鼠海马CA3区锥体细胞层细胞排列相对整齐、紧密，细胞计数明显增多（p=0.039）。SA-β-GAL在CR大鼠的神经元染色微弱（p＜0.001）。18月龄和24月龄的大鼠海马组织的SA-β-GAL染色较6月龄增加（P＜0.001），培养12天和20天的海马神经元SA-β-GAL染色较培养6天的海马神经元明显增多（P＜0.001）。结论：1．对老年早期的大鼠给予6个月的限制饮食（CR）可以提高大鼠的生存率。2．CR能够提高大鼠的活动能力，反映了大鼠的健康状况改善。3．CR延缓衰老引起的空间学习能力减弱，提高参考记忆力的准确性。4．大鼠空间学习记忆能力的提高可能与CR延缓了大鼠海马CA3区神经元的衰老有关。5．SA-β-GAL是可靠的检测海马CA3区海马衰老神经元的生物学标记物。限制饮食（calorie restriction，CR）能够不同程度的延长多种生物的寿命，有效延缓的衰老和减少老年相关疾病的发生。了解CR引起这些效应的作用机制有助于指导人类的饮食和生活方式，起到延缓衰老，预防疾病发生的作用。然而，CR是如何发挥抗衰老的作用，参与其调节的分子机制还远未了解清楚。沉默信息调节因子2（silence information regulator 2，Sir2）是一种烟酰胺腺嘌呤核苷酸（nicotinamide adenine dinucleotide，NAD）依赖性去乙酰化酶，研究发现，在CR能够引起Sir2的上调，并且这一现象从低等生物酵母到高等生物如哺乳类动物，灵长类动物等普遍存在，因此，Sir2被认为是介导CR发挥效应的一个关键调节因子。哺乳动物的同源基因（sirtuin 1，SirT1）能够与多种底物作用，如p53，NF-kappaB和叉头框转录因子（Forkhead transcription factors，FoxO3a）参与细胞对氧化应激的抵抗力和基因稳定性的调节。在哺乳动物生长激素和IGF-1也参与了寿命调节，对于信号通路中FOXO家族的一员，FoxO3a参与调节多种生物学功能，包括细胞周期停滞，修复受损的DNA和衰老。CR能够在多种生物中引起胰岛素／胰岛素样生长因子-1（insulin／insulin-like growth factor 1，insulin／IGF-1）信号的抑制，其中FOXO家族是这个信号通路下游的一个主要靶点。因此，FoxO3a在CR调节中可能参与了重要的介导作用，同时FoxO3a在大脑中大量表达，尤其在发育和成年的海马，这提示FoxO3a可能参与了学习记忆的过程和（或）调节神经元的衰老。但对于FoxO3a在CR的大鼠海马的表达变化及与SirT1变化的关系，仍是一个值得探讨的问题。为探讨限制饮食引起的延缓神经元衰老的调节机制，本研究选用结合观察SirT1及FoxO3a及相关信号因子，同时，利用SirT1的激动剂白藜芦醇作用于PC12细胞，观察对培养细胞的生物学和分子生物学改变，进一步探讨SirT1与FoxO3参与CR的调控机制。材料与方法：实验动物选用18月龄的健康雄性Sprague Dawley（SD）大鼠，根据进食情况分为两组，一组为CR组（n=29）给予60％的进食量，另一组为AL组（n=31）自由进食，采用免疫组织化学和Westem blot方法观察海马CA3区SirT1和FoxO3a蛋白水平的改变。在培养的PC12细胞中，给予不同浓度的白藜芦醇，观察细胞形态学改变，利用MTT试验观察细胞增殖情况，利用RT—PCR观察SirT1，FoxO3a及细胞周期素依赖性激酶（Cyclin-dependent Kinase Inhibitors，Cdk）抑制蛋白p27^KIP1的mRNA水平的改变，利用流式细胞术观察白藜芦醇对PC12细胞周期的影响。结果：免疫组织化学和Westem blot检测SirT1的表达结果显示，CR组明显升高（p=0.018），免疫组织化学观察到FoxO3a在两组中呈现细胞定位的不同，CR组FoxO3a在细胞核内的表达减少（p=0.002）而转移至胞浆（p=0.005）。在培养的PC12细胞中，随着白藜芦醇浓度增加（5-50μmol／L）出现明显的细胞生长抑制（p＜0.001），在细胞周期的检测中，发现10μmol／L的白藜芦醇使G0／G1期的细胞比例增加（P=0.023）。RT-PCR结果显示，10μmol／L的白藜芦醇能够使SirT1表达升高（p=0.02），而p27^KIP1在20μmol／L和50μmol／L白藜芦醇刺激下表达升高（p＜0.001），而FoxO3a的改变不明显（p=0.09）。结论：1．在SirT1的上调和FoxO3a的失活参与了限制饮食延缓海马神经元衰老的调节2．SirT1的激活能够使PC12细胞G0／G1期停滞更多还原

【Abstract】 Background and objectiveCalorie Restriction （CR） is one of the most effective methods to increase longevity,delay ageing and improve health. CR retards age-related neurondegeneration, which ischaracterized by decline in learning and memory ability. Majority of former data wereobtained from life-long CR rats or mice. Whereas there are numerous reports aboutage-related diseases prevention by CR, the effects of CR on cognitive performanceare largely unknown. We assumed that the maintained learning and memory ability inCR rat was related to the postponed neural senescence in hippocampus, where is oneof the most important regions involved in learning and memory. As a senescencemarker, senescence associatedβ-galactosidase （SA-β-GAL） has been widely adoptedin replicative senescence in vitro; however, the reliability of this biomarker in braintissue has not been well understood. In this investigation, we applied a shorter periodof the treatment of CR from early old age rats and evaluated the survival and learningand memory ability by behavioral study. We applied SA-β-GAL to detect senescentneurons in hippocampus to examine the senescence status of hippocampal neurons ofCR rat. Meanwhile, we investigated the activity of SA-β-GAL in rat hippocampus atdifferent ages and in prolong cultured hippocampal neurons, as a parallel study to testthe marker’s reliability.Materials and methodsMale Sprague-Dawley （SD） rats with age of 18 month were subjected to restrictedintake in CR group （n=29） by 60％comparing with that of rats fed ad libitum （AL）（n=31） for 6 months. We compared the overall health status, including survival andlocomotor activity by open-field test. Spatial learning and reference memory ability ofthe rats were evaluated by Morris Water Maze （MWM）. Cell numbers in CA3 region of hippocampus were counted after Nissl staining. Hippocampus of 6 Mon （n=10）, 18Mon （n=6） and 24 Mon （n=7） SD rats were observed by histochemical staining forSA-13-GAL. Prolonged cultured primary hippocampal neurons of SD rat wereobserved by cytochemical staining for SA-13-GAL.ResultsGrowth rate of the CR rats demonstrated a period of decline during the first 10weeks of treatment, and thereafter became steady below that of AL rats （p＜0.001）.The CR rats showed a higher survival （p=0.039）. In the open-field test, the CR ratstraveled longer （p=0.021） especially at the centre place （p=0.048）, and moved faster（p=0.021） as well as in circumjacent area （p=0.012） than the AL rats. The meanescape latency （EL） of CR group was shorter than that ofAL group （p=0.038） innavigation trials of MWM. The differences in the times crossing the former annualand percentage of time and distance covered in the target quarterⅢdidn’t reach thestatistic significance（p=0.324 and p=0.367）, however, great difference showed in thatof quarterⅡ（p=0.027and p=0.029）. In the histological study, CR rats showed tighterpyramidal cells line and higher neuron number in the CA3 region in hippocampusthan that of AL group in Nissl staining （p=0.039）. The staining of SA-β-GAL wasweaker in pyramidal cells of hippocampus of CR rats than in AL rats （p＜0.001）.SA-β-GAL activity elevated in hippocampal pyramidal cells of CA3 region withadvancing age （p＜0.001） and with culture period of hippocampal neurons （p＜0.001）.Conclusion:1. The 6-month treatment of 60％CR elevated survival of early old age rat.2. The 6-month treatment of 60％CR benefited health status by locomotor activity.3. The 6-month treatment of 60％Calorie Restriction delayed the age-relatedinability to learn and improved the accuracy of the memory of early old rats.4. A Calorie Restriction regimen for early old rats delayed the senescence ofhippocampal neurons may contribute to the better learning and memoryperformance.5. SA-β-GAL was a reliable biomarker to detect senescent cells in CA3 region of hippocampus. Background and Objective:It has been known that calorie restriction （CR） can extends median and maximallife span in a wide spectrum of organisms and retard or prevent age-dependentdeterioration of brain. However the molecular mechanisms of the benefit of CR arestill largely unknown. An understanding of the molecular mechanisms that underliethe beneficial effects of CR will help identify novel dietary, and lifestyle strategies forslowing the rate of aging and preventing these diseases.Studies show that the elevation of silence information regulator 2 （Sir2）, anicotinamide adenine dinucleotide （NAD）-dependent deacetylase is one of thecommon effects of CR in a wide spectrum of organisms. It has been postulated thatthe mammalian homolog sirtuin 1 （SirT1） is one of the key regulators of thedownstream effects of CR. SifT1 posses a large list of substrates, including p53,NF-kappaB and forkhead transcription factors （FoxO3a）, which modulate cellularresistance to oxidative and genotoxic stress.As one of the members in mammalian FOXO families, FoxO3a is involved incontrolling various biological functions, such as cell cycle arrest, repair of damagedDNA, and senescence. FOXO family is one of the main down-steam targets ofinsulin/IGF-1 pathway, which is another way for CR to extend life-span. In mammals,growth hormone （GH） and insulin-like growth factor 1 （IGF-1） appear to be critical indetermining lifespan. FoxO3a is highly expressed in brain especially in thedeveloping and adult hippocampus, indicating FoxO3a might take a role in memoryformation and modulate neuronal senescence.Both SirT1 and FoxO3a mediate the important pathway for neuronal growth and physiology. However their modulations in CR rats are still unclear. To elucidate thecontribution of both factors to the effects of CR is helpful to understand theunderlining mechanism. We studied the expression of SirT1 and FoxO3a inhippocampus of CR rats. Furthermore, we applied resveratrol as SirT1 activator toPC12 cells to examine the cell cycle to explore the potential interaction with FoxO3a.Materials and methods:18-month rats were subjected to restricted intake by 60％comparing with that ofrats fed ad libitum （AL） for 6 months. In addition, we examined the expression ofSirT1 and FoxO3a, which mediate important pathways for neuronal growth,senescence and determine the fate of neurons, to explore underlining mechanism ofeffect of CR on aging neurons, using immunohistochemistry and western blot. PC12Cells were analyzed by MTT assay to determine the resveratrol sensitivity at differentconcentration （5, 10, 20, 50μmol/L）. SirT1, FoxO3a and p27^KIP1 were measured byRT-PCR after PC 12 cells pretreated with resveratrol at different concentration for 30min. The distribution of cells in the cell cycle was determined by flow cytometryusing propidium iodide-stained nuclei.Results:The level of SirT1 expression was enhanced both in immunohistochemistry（p=0.018） and Western blot （p=0.02）. The cytoplasm location of FoxO3a increased inthe hippocampal neurons of the CR rats （p=0.005） showed by immunohistochemistry.In PC12 cell, with increased dose （5-50μmol/L）, RSV showed a significant inhibitionin cell proliferation by MTT assay and morphological observation （p＜0.001）. Thepercentage of cells in G0/G1 phase tested by flow cytometer increased at the10μmol/L dose of RSV （p=0.023）. RT-PCR analysis demonstrated that SirT1 elevatedat the concentration of 10μmol/L RSV （p=0.02）. p27^KIP1 elevated with the increaseconcentration of RSV（p＜0.001）. The changes of the levels of FoxO3a mRNA didn’treach the statistical significant level （p=0.09）.Conclusion:1. Both the unregulated SirT1 and suppressed FoxO3a may participate in postponing the senescence of hippocampal neurons in CR rats.2. SirT1 could be activated by Resveratrol at the concentration of 10μmol/L inPC12 and leads to cell cycle G0/G1 phase arrest.更多还原