【作者】 杨敬华；

【导师】 蔡原；

【作者基本信息】 中国医科大学 ， 劳动卫生与环境卫生学， 2009， 博士

【摘要】 目的稀土元素（rare earth elements,REEs）是一组化学性质相近的镧系元素:镧、铈、镨、钕、钷、钐、铕、钆、铽、镝、钬、铒、铥、镱、镥以及与镧系元素性质相近的钪和钇。近十几年来,稀土技术在我国迅速推广和应用,涉及农业、畜牧业、水产养殖、工业和医药等多个领域,特别是稀土微肥、微饲被广泛应用于农业、畜牧业生产。随着应用的日益广泛,越来越多的REEs进入环境,并通过食物链的作用进入人体,REEs的环境残留、蓄积及其对人体健康的影响成为不容忽视的卫生学问题,因此REEs的环境影响和健康效应值得深入研究。研究表明,REEs对机体具有Hormesis效应,低剂量时对生物体具有促进作用,高剂量时则影响机体的脑、肝、肾、心脏等多个组织和系统。人群调查显示,稀土矿区的儿章在智商均数、学习记忆等认知能力方面均较对照区儿童明显低下。动物实验表明,REEs可以造成受试动物的神经行为、学习记忆明显受损。REEs对中枢神经系统的影响日益受到重视。镧属于轻REEs,化学性质极为活泼,几乎能与所有的元素和组织成分发生反应。镧也是自然界中含量较多的REEs,且在脑中的蓄积性较强,常作为REEs神经毒性研究的代表物质。目前镧乃至REEs损害学习记忆的机制尚不明确。海马是与学习记忆有关的关键脑区,Ca²⁺/CaM-CaMKIV-CREB通路在学习记忆的信号传导中发挥重要作用,但其在镧的作用下有无变化尚未见报道。本研究通过对大鼠在出生至断乳后一个月期间进行氯化镧（lanthanumchloride,LaCl₃）染毒,建立亚慢性LaCl₃暴露致学习记忆能力受损的动物模型,运用神经行为学、生物化学、分子生物学和免疫组织化学等多种技术手段,明确镧对Ca2+/CaM-CaMKIV-CREB通路的影响及该通路各主要成分的变化与镧致突触可塑性、神经行为学改变之间的关系,从分子水平探讨镧损害学习记忆能力的信号转导机制,为深入阐明镧乃至REEs的神经毒作用机制、有效干预镧乃至REEs所致智力损害和认知功能缺陷提供基础资料与科学参考。方法由中国医科大学实验动物中心提供的实验用Wistar大鼠60只,体重260±10g,雌雄比例2:1。实验动物室温度17～23℃,相应湿度45～55%,动物饲料由实验动物中心提供。随机将雌鼠分为对照组和低、中、高剂量LaCl₃组,然后将雌雄大鼠按2:1同笼进行交配,次日晨发现阴栓或阴道分泌物镜检有精子者定为受孕,记为妊娠第0天。对照组孕鼠在产仔后饮用蒸馏水,低、中、高剂量LaCl₃组孕鼠在产仔后分别饮用0.25%、0.5%、1.0%LaCl₃蒸馏水溶液。各染镧组仔鼠在断乳前经由吸吮母乳染镧,断乳后则通过自行饮用0.25%、0.5%和1.0%LaCl₃蒸馏水溶液染镧,至断乳后1个月结束,然后进行各项指标测定。采用电感耦合等离子体质谱法测定仔鼠海马组织镧含量,采用水迷宫、穿梭箱和跳台试验三种神经行为学测试方法测定仔鼠的学习记忆能力,采用尼氏染色观察仔鼠海马神经细胞中尼氏体表达情况,应用透射电子显微镜技术观察仔鼠海马神经元和神经突触超微结构,采用邻苯二醛柱前衍生高效液相色谱法检测仔鼠海马兴奋性氨基酸（谷氨酸和天冬氨酸）含量,采用Fura-2荧光探针法检测仔鼠海马细胞内Ca²⁺浓度,采用磷酸二酯酶法测定仔鼠海马CaM活性,采用Western blot测定仔鼠海马p-CaMKIV和p-CREB的蛋白表达水平,采用RT-PCR法测定仔鼠海马c-fos和BDNF mRNA表达水平,采用免疫组织化学法测定仔鼠海马c-fos蛋白表达水平。结果1、氯化镧对大鼠学习记忆能力和海马超微结构的影响LaCl₃组仔鼠海马中镧含量显著高于对照组。随着染镧剂量的增加,镧在仔鼠海马中的蓄积量随之增加。水迷宫测试时,低、中剂量LaCl₃组仔鼠错误次数和潜伏期显著高于对照组,高剂量LaCl₃组仔鼠错误次数和潜伏期显著高于对照组和低剂量LaCl₃组。穿梭箱测试时,中剂量LaCl₃组仔鼠电击次数、电击时间和主动逃避潜伏时间显著多于对照组,高剂量LaCl₃组仔鼠电击次数、电击时间和主动逃避潜伏时间显著多于对照组和低剂量LaCl₃组。跳台测试时,低剂量LaCl₃组仔鼠潜伏期显著少于对照组;中剂量LaCl₃组仔鼠错误次数显著多于对照组,潜伏期显著少于对照组和低剂量LaCl₃组;高剂量LaCl₃组仔鼠错误次数显著多于对照组和低剂量LaCl₃组,潜伏期显著少于对照组和低、中剂量LaCl₃组。尼氏染色显示,LaCl₃染毒组仔鼠海马CA1、CA3和DG区神经细胞中尼氏体表达水平显著低于对照水平,而且随着LaCl₃暴露水平的增加,仔鼠海马CA1、CA3和DG区神经细胞中尼氏体表达进一步减弱,高剂量LaCl₃组部分细胞中尼氏体消失,出现空泡变性。此外,电子显微镜观察显示,LaCl₃染毒组仔鼠海马神经元超微结构受到损害,而且神经突触超微结构发生改变,突触活性带较短、突触后致密物较薄以及突触界面曲率减小。2、氯化镧对大鼠海马兴奋性氨基酸、Ca²⁺/CaM和CaMKIV的影响低剂量LaCl₃组仔鼠海马谷氨酸含量、细胞内Ca²⁺浓度显著高于对照组,而CaM活性、p-CaMKⅣ蛋白表达水平显著低于对照组;中剂量LaCl₃组仔鼠海马谷氨酸含量、细胞内Ca²⁺浓度显著高于对照组,而CaM活性显著低于对照组、p-CaMKⅣ蛋白表达水平显著低于对照组和低剂量LaCl₃组;高剂量LaCl₃组仔鼠海马谷氨酸含量、细胞内Ca²⁺浓度显著高于对照组和低、中剂量LaCl₃组,天冬氨酸含量显著高于对照组和低剂量LaCl₃组,而CaM活性显著低于对照组和低剂量LaCl₃组、p-CaMKⅣ蛋白表达水平显著低于对照组和低、中剂量LaCl₃组。3、氯化镧对大鼠海马CREB、BDNF和c-fos表达的影响低、中剂量LaCl₃组仔鼠海马p-CREB蛋白、c-fos mRNA和c-Fos蛋白表达水平显著低于对照组,高剂量LaCl₃组仔鼠海马p-CREB蛋白、c-fos mRNA和DG区c-Fos蛋白表达水平显著低于对照组和低、中剂量LaCl₃组,CA1和CA3区c-Fos蛋白表达水平显著低于对照组和低剂量LaCl₃组。低剂量LaCl₃组仔鼠海马BDNFmRNA表达水平与对照组无显著性差异,中、高剂量LaCl₃组海马BDNF mRNA表达水平显著低于对照组和低剂量LaCl₃组。结论1、亚慢性LaCl₃暴露造成大鼠的学习记忆能力低下,海马神经元和神经突触的超微结构受到损害。2、亚慢性LaCl₃暴露引起大鼠海马兴奋性氨基酸含量异常、钙稳态失衡、CaM活性和CaMKIV磷酸化水平降低。3、亚慢性LaCl₃暴露导致大鼠海马CREB磷酸化水平降低,BDNF和c-fos的表达水平下降。4、亚慢性LaCl₃暴露损害大鼠的学习记忆能力,其机制可能与海马内兴奋性氨基酸含量异常、钙稳态失衡、CaM活性下降以及随后的CaMKIV-CREB信号转导途径表达下调有关。更多还原

【Abstract】 ObjectiveRare earth elements（REEs） include lanthanum（La）,cerium,praseodymium, neodymium,promethium,samarium,europium,gadolinium,terbium,dysprosium, holmium,erbium,thulium,ytterbium,lutetium,scandium and yttrium in the periodic table of elements.In recent years,rare-earth techniques have been applicated widely in many fields such as agriculture,farming,aquaculture,industry and medicine in China. In particular,trace fertilizer and feedstuff containing REEs are used widespread in agriculture and farming.With widespread applications,more and more rare-earth REEs enter into the environment and accumulate in human body by food chain.Residue, accumulation of REEs in the environment and their effects on human health should not be ignored.Therefore,the effects of REEs on the environment and human health deserve further investigation.It is shown that REEs have hormesis effects.That means that low-level REEs can promote the growth of organisms,but high-level REEs have adverse effects on many tissues and organs such as brain,liver,kidney,heart and so on.Surveys on population have reported that average intelligence quotients,cognitive functions such as learning and memory of children living in the REEs-high regions were significantly lower than those of control children.Experimental studies have also shown REEs could significantly impair neurological behavior,learning and memory of animals.Nowadays, effects of REEs on central nervous system have been paid more attention.La is a light REE with active chemical properties,and it can react with almost all elements and components of tissues.Among REEs,La is also abundant in the environment. Furthermore,there is more accumulation of La in the brain.Therefore,La is often used as a representative to probe into the neurological adverse effects of REEs.Nowadays, mechanism underlying the learning and memory impairments induced by La is still not clear.Hippocampus is the key brain region related with learning and memory. Ca2+/CaM-CaMKIV-CREB pathway plays an important role in signal transduction associated with learning and memory.However,there is no reports that whether there are changes in this signal pathway induced by La or not.Animal model of learning and memory impairment was made in this study by subchronic administration of lanthanum chloride（LaCl₃） to rats from birth to weaning for one month.By neuroethological,biochemical,molecular biological and immunohistochemical methods and techniques,the effects of La on Ca2+/CaM-CaMKIV-CREB signal pathway were investigated,and the relationship between main components in signal pathway and changes in synaptic plasticity and neuroethological performances were discussed.The investigation of signal transduction mechanism underlying learning and memory impairments induced by La from molecular level could provide basic data and scientific reference for further elucidation of neurotoxic mechanism of La and even REEs.Based on these data,intelligence impairment and cognition deficiency caused by La and even REEs could also be prevented effectively.MethodsSixty Wistar rats weighing（260±10）g were obtained from the Center for Experimental Animals of China Medical University.The ratio of female to male is 2. Rats were housed at the standard laboratory condition with environmental temperature of（17～23）℃and humidity of 45～55%.The food was provided by Center for Experimental Animals of China Medical University.Animals were observed for 7 days before mating.Female rats were divided randomly into four groups:control,low-, middle- and high-dose LaCl₃ groups.Female and male rats were mated（female:male= 2:1）.Occurrence of copulatory plug or sperm in the vaginal discharge in the second day after mating was used to indicate successful mating and day 0 of pregnancy.During the period of lactation（3 weeks）,the control lactational rats drinked distilled water,and lactational rats in low-,middle- and high-dose LaCl₃ groups were exposed to LaCl₃ （99.9%,China） in distilled drinking water under one of three doses（0.25%,0.5%,and 1.0%）.The pups were firstly exposed to LaCl₃ by parental lactation for postnatal 3 weeks and then orally administrated with 0.25%,0.5%,and 1.0%LaCl₃ in distilled drinking water for 1 month,respectively.La contents in the hippocampus of pups were determined by inductively coupled plasma mass spectrometry.Learning and memory of pups were measured by neuroethological tests including water maze,shuttle box and jumping stand tests.The expression of Nissl body in the hippocampus of pups was observed by Nissl staining.The ultra-structural features of neurons,synapses in the hippocampus of pups were observed with a transmission electron microscope.The contents of excitatory amino acid such as glutamic acid and aspartic acid in the hippocampus of pups were determined by o-phthalaldehyde precolumn derivatization with high performance liquid chromatography.The concentrations of intracellular calcium ions in the hippocampus of pups were detected by Fura-2 fluorescent probe. The activities of calmodulin（CAM） in the hippocampus of pups were determined by phosphodiesterase（PDE）.The phosphorylated levels of calmodulin dependent protein kinaseⅣ（CaMKIV） and cAMP response element binding protein（CREB） in the hippocampus of pups were determined by western blot.The mRNA expression levels of c-fos and BDNF in the hippocampus of pups were determined by reverse transcription polymerase chain reaction（RT-PCR）.The protein expression levels of c-Fos in the hippocampus of pups were measured by immunohistochemistry.Results1、Effects of LaCl₃ on learning,memory and ultra-structure in the hippocampus of ratsLa contents in the hippocampus of LaCl₃-administrated pups were significantly higher than those of control group.With LaCl₃-administrated dose increasing,La accumulation in the hippocampus of pups increased.In the water maze test,wrong number and latency of low- and middle-dose LaCl₃ groups were significantly higher than that of control group,and wrong number and latency of high-dose LaCl₃ groups were significantly increased as compared with than that of control and low-dose LaCl₃ groups.In the shuttle box test,shock number,time and latency of active avoidance in shock stimulation of middle-dose LaCl₃ groups were significantly higher than that of control group,and these parameters of high-dose LaCl₃ groups were significantly increased as compared with than that of control and low-dose LaCl₃ groups.In the jumping stand test,latency of low-dose LaCl₃ group was significantly lower than that of control group.As to middle-dose LaCl₃ group,wrong number was significantly increased as compared with than that of control group,but latency was significantly lower than that of control and low-dose LaCl₃ groups.As to high-dose LaCl₃ group, wrong number was significantly increased as compared with than that of control and low-dose LaCl₃ groups,but latency was significantly lower than that of control,low-and middle-dose LaCl₃ groups.The expression of Nissl body in CA1、CA3 and DG area of neurons in the hippocampus of LaCl₃ groups were lower significantly than those of control group.The expression of Nissl body in CA1、CA3 and DG area of neurons in the hippocampus of pups decreased further with administrated-dose increasing. Furthermore,Nissl body in CA1、CA3 and DG area of some neurons in the hippocampus of high-dose LaCl₃ group disappeared,and there was vacuolar degeneration. Besides,it was shown in this study that there were damages in the ultra-structures of neurons in the hippocampus of LaCl₃-administrated groups.Besides,there were ultra-structure changes in the synapses of hippocampus in LaCl₃-administrated pups. These ultra-structure changes included short active zone,small synaptic curvature and thin post-synaptic density.2、Effects of LaCl₃ on excitatory amino acids,Ca²⁺/CaM and CaMKIV in the hippocampus of ratsThe contents of glutamic acid and concentration of intracellular calcium ions in the hippocampus of low-dose LaCl₃ group were significantly higher than those of control group,but the activities of CaM and expression levels of p-CaMKIV were significantly decreased as compared with those of control group.The contents of glutamic acid and concentration of intracellular calcium ions in the hippocampus of middle-dose LaCl₃ group were significantly higher than those of control group,but the activities of CaM were significantly decreased as compared with those of control group, and the expression levels of p-CaMKⅣwere significantly lower than those of control and low-dose LaCl₃ groups.As to high-dose LaCl₃ group,the contents of glutamic acid and concentration of intracellular calcium ions in the hippocampus were significantly higher than those of control,low- and middle-dose LaCl₃ groups,and the contents of aspartic acid in the hippocampus were significantly increased as compared with control and low-dose LaCl₃ groups.However,the activities of CaM of high-dose LaCl₃ group were significantly lower than those of control and low-dose LaCl₃ groups,and the expression levels of p-CaMKIV were significantly lower than those of control,low-and middle-dose LaCl₃ groups.3、Effects of LaCl₃ on CREB,BDNF and c-fos expression in the hippocampus of ratsThe expression levels of p-CREB,c-fos mRNA and c-Fos in the hippocampus of low- and middle-dose LaCl₃ group were significantly lower than those of control group. The expression levels of p-CREB,c-fos mRNA of the hippocampus and c-Fos in DG area of the hippocampus in high-dose LaCl₃ group were significantly lower than those of control,low- and middle- dose LaCl₃ groups.The expression levels of c-Fos protein in CA1,CA3 area of the hippocampus in high-dose LaCl₃ group were significantly lower than those of control and low-dose LaCl₃ groups.As to the expression levels of BDNF mRNA of the hippocampus,there were no differences between control and low-dose LaCl₃ group.The expression levels of BDNF mRNA in the hippocampus of middle-,and high-dose LaCl₃ group were significantly lower than those of control and low-dose LaCl₃ groups.Conclusion1、Subchronic exposure to LaCl₃ could lead to learning and memory impairment and damages in ultra-structures of neurons and synapses in the hippocampus of rats.2、Subchronic exposure to LaCl₃ could induce change in the contents of excitatory amino acids and disturbance of calcium homeostasis,decrease the activities of CaM and the phosphorylated levels of CaMKIV in the hippocampus of rats.3、Subchronic exposure to LaCl₃ could cause lower levels of phosphorylated CREB,decrease the expression level of c-fos and BDNF in the hippocampus of rats.4、Subchronic exposure to LaCl₃ did harm to learning and memory of rats,which is possibly related to change in the contents of excitatory amino acids,disturbance of calcium homeostasis,decrease in the activities of CaM and consequent down-regulation of CaMKⅣ-CREB signal transduction pathway in the hippocampus of rats.更多还原