【作者】 祝文静；

【导师】 章子贵；

【作者基本信息】 浙江师范大学 ， 动物学， 2010， 硕士

【摘要】 氟虽是人体必需的微量元素之一,但长期过量摄入会引起慢性氟中毒(Chonic fluorosis)。氟对中枢神经系统的毒性表现为影响儿童智力发育,抑制自发性神经活动,损害学习记忆能力等。海马是与学习记忆密切联系的关键脑区,也是氟神经毒作用的靶部位之一。已有研究证明,过量氟会损伤海马内的突触超微结构,但鲜见氟影响突触相关蛋白表达的报道。突触后致密物蛋白95(postsynaptic density95, PSD-95)是突触后致密物的主要蛋白之一,本身并无活性,N-甲基-D-天(门)冬氨酸(N-methyl-D-aspartate, NMDA)受体则是与学习记忆相关的重要蛋白。PSD-95可以通过不同的结构域串集NMDA受体其相关分子,形成信号复合物,在突触水平转导并整合兴奋性信号。在突触可塑性过程中,NMDA受体起着极为关键的作用。我们推测,PSD-95表达水平的变化可能会影响NMDA受体的簇集,进而影响动物的学习记忆能力。自由基损伤学说被公认为氟中毒发病机制之一。该学说认为,正常情况下机体内由于各种抗氧化酶(如超氧化物歧化酶superoxide dismutase, SOD)和抗氧化物质(如维生素C)的存在,自由基的产生和清除处于动态平衡状态。氟中毒发生时,体内脂质过氧化作用增强,氧化系统与抗氧化系统的平衡遭到破坏,从而导致脂质过氧化产物丙二醛(malondialdehyde, MDA)含量增加。SOD、MDA是目前检测脂质过氧化水平的常用指标,但由于受多种因素影响,特异性与敏感性不强。因此,本实验通过复制慢性氟中毒动物模型,宏观上观察慢性氟中毒对大鼠脑功能的影响,微观上选用特异性与敏感性较好的自旋标记法检测脑内氧化应激水平,并采用免疫组化法(immuno-histochemical stain, IHC)检测海马CA3区PSD-95蛋白的表达水平。通过研究,拟筛选出脑内学习记忆相关脑区(海马)氟中毒相关蛋白和因子(靶分子),为深入探讨氟对中枢神经系统毒性作用的分子生物学机制提供一定的理论基础,为氟中毒的早期诊断和早期治疗提供科学依据,从而拓宽为我省氟污染区饮水型地氟病患者制定合理的综合治疗方案的思路。研究方法：192只初断乳的雄性SD大鼠随机分为四组,即对照组(饮用自来水,水氟含量低于0.5mg／L),低氟组(饮水含氟化钠(NaF)15mg／L),中氟组(饮水含NaF30mg／L),高氟组(饮水含NaF 60mg／L)。各组以配制的溶液作为饮水唯一来源,自由摄食、饮水,饲养时间为18个月。实验中每三个月对动物进行行为检测,以开场行为检测大鼠的自发活动和探究行为,以水迷宫检测大鼠的空间学习记忆能力；分别在染氟中期(9个月)和染氟结束后(18个月)分两批断头处死大鼠,HE染色作海马CA3区病理学观察,自旋标记法检测突触体膜流动性的变化,免疫组化法检测海马CA3区PSD-95蛋白表达的改变。实验结果：1、开场行为结果显示,慢性氟中毒可抑制大鼠在新异环境中的自发活动,使跑动格数减少(p<0.05),站立频率降低(p<0.05或p<0.O1)。水迷宫结果显示,各染氟组大鼠搜索平台潜伏期、搜索平台总路程普遍高于对照组,说明慢性氟中毒大鼠空间学习记忆能力下降。此外,染氟浓度越大、染毒时间越长,氟的这种毒性效应越明显。2、突触体膜流动性的检测结果显示,随着染氟浓度的增加,16-DSA标记的突触体膜序参数值与旋转相关时间呈递增趋势(p<0.05或p<0.01),即海马突触体膜流动性逐渐降低。说明慢性氟中毒可导致细胞膜流动性的改变,染氟18个月组大鼠的膜流动性变化比染氟9个月组更显著。值得注意的是,染氟9个月高氟组大鼠海马突触体膜序参数、旋转相关时间不升反降,基本回到低氟组的水平。3、HE染色结果显示,慢性氟中毒大鼠海马锥体细胞细胞数量减少,细胞轮廓消失,细胞出现空泡等。IHC结果显示,随着染氟浓度的增加,PSD-95的表达水平呈递减趋势,与同龄对照组相比,p<0.05或p<0.01。提示慢性氟中毒可损伤海马神经细胞,下调突触后致密物重要蛋白PSD-95的表达水平,影响NMDA等受体复合物的簇集,使学习记忆能力下降。4、在正常衰老过程中,大鼠存在增龄性的自发活动减少,学习记忆能力降低,膜流动性下降的现象。而染不同浓度氟后加剧了这种趋势,可见动物的学习记忆行为受多因素影响,衰老与环境因素(慢性氟中毒)致氧化应激均可在宏观上引起动物学习记忆损伤,微观上影响突触体膜流动性的降低和PSD-95表达水平的下降。综上所述,饮水型慢性氟中毒可抑制大鼠的自发性神经活动,损害空间分辨学习记忆能力；而氟可透过“血-脑”屏障,攻击神经细胞膜,导致突触体膜流动性降低,引起突触后致密物蛋白PSD-95表达的减少,这可能是饮水型慢性氟中毒致脑损伤的分子机理之一。根据自由基损伤学说,氧化应激可能参与了氟的这一系列毒性效应。鉴于PSD-95对于慢性氟中毒的敏感性,可以进一步研究动物发育早期氟化物对PSD-95表达水平的影响,为筛选脑内氟致脑损伤的生物标志物研究提供理论基础。更多还原

【Abstract】 Fluoride is an essential trace element for all mammalian species, but meanwhile its excess is known to be toxic both to human and animals, such as affecting development of children’s intelligence, impairing learning and memory ability and so on. Research shows that hippocampus is a target site of fluoride, which is closely related to spatial learning and memory ability. Our previous research indicated that fluoride impairs the synaptic ultra microstructure in rat hippocampus, but the concrete mechanism is still unknown. Post synaptic density 95(PSD-95) is one of the main proteins in post synaptic density; though it has no activity, it can connect NMDA receptor and other related molecules to form a signal-complex and combine the excitatory signal-transduction in synaptic level through its different domains. NMDA receptor plays a very important role in forming the synaptic plasticity. Above all, we presumed that changes of PSD-95 expression level in rat hippocampus can effect the clustering of NMDA receptor and then effects learning abilitiy of animals.A variety of mechanisms have been proposed to explain fluoride-induced toxicity, including free-radical theory. It is considered that fluorosis could enhance the lipid peroxidation in vivo, break the balance between oxidation system and antioxidant system, and change the content of superoxide dismutase (SOD) and malondialdehyde (MDA). SOD and MDA content are usually regarded as one of index to lipid peroxidation level, but their specificity and sensitivity are not good enough because of variety reasons. Thus, we chose rat model of chronic fluorosis to explore the effects of different doses of fluoride on spatial learning and memory firstly on macroscopic level, and then detected synaptic membrane fluidity and expression levels of PSD-95 on microcosmic level, to offer some useful clues for further study of fluoride-induced neurotoxicity and provide a scientific basis for early diagnosis and treatment of fluorosis.Methods:192, just weaning male Sprague-Dawley(SD) rats (64±8.8g) were randomly divided into four groups and given 15,30,60 mg/L NaF solution and distilled water respectively for 9 and 18 months. Rats were fed standard diet throughout the experiment and water was given ad libitum. Animal’s behaviors were tested every three months, including locomotor activity and exploratory behavior by open field test, learning and memory ability by morris water maze test; animals were thereafter decapitated when exposure was stopped, then evaluated pathological change in hippocampus with HE staining, membrane fluidity with ESR method, and expression level of PSD-95 in hippocampus CA3 region with IHC method.Results:1. Open field test reveal that chronic fluorosis suppressed rat’s locomotor activity and exploratory behavior in extraneous environment, such as reduced locomotion and kearing frequency (p<0.05, p<0.01). The results of morris water maze test showed that mean escape latency and escape length of rat drinking fluoride water was higher when compared with control group. It indicated that chronic fluorosis impaired rat’s spatial learning and memory ability. In addition, fluoride toxicity is connected with both fluoride concentration and time. Fluoride concen-tration is higher and exposure time is longer, the toxicity of fluoride may be stronger.2. The results of synaptic membrane fluidity showed that, order parameter(S) and rotational correlation time of synaptic membrane which marked by 16-DSA increased as fluoride concentration increased, which means the hippocampus synaptic membrane fluidity decreased in some degree. There was something need to be noticed that rotational correlation time of rat in 9-month old high-fluoride group decreased insteadly, almost decreased to the level of that in low-fluoride group.3. Chronic fluorosis could impair rat brain nerve cells. Results of HE staining showed that, the distribution of rat hippocampus pyramidal cells were sparse, cell outline disappeared. Result of IHC staning showed that, the expression level of protein PSD-95 decreased as fluoride concentration increasing (p<0.01,p<0.05). It is suggested that chronic would impair nerve cell, down-regulated expression of PSD-95 protein and impacted on aggregation of NMDA receptor complex, resulting in learning and memory ability decline finally.4. Brain aging can decrease rat locomoter activity, spatial learning and memory ability, and membrane fluidity. This research showed that group of 18-month old rat escape latency was significant different (p<0.01) when compared with group of 3-month old rat, and this trend intensified with the increase of fluoride concentration. So we can learn from it that animals’learning abilities and memories can be affected by several factors, including senility and environmental factors as chronic fluorosis.Above all, chronic fluorosis can depress rat locomoter activity and impair spatial learning and memory ability. And excessive fluoride can pass blood-brain barrier, attack the nervous cells membrane, decrease rat hippocampus synaptic membrane fluidity and expression level of post synaptic density protein PSD-95. According to free-radical theory, oxidative stress may be a resource of fluoride toxicity. Since the expression level of PSD-95 is sensitive to chronic fluorosis, we could make further research of fluoride effect in early development stage, and it can offer us a theoretical basis in research of PSD-95 as an environmental neurotoxic substance biomarker.更多还原