【作者】 梁瑞峰；

【导师】 牛侨；

【作者基本信息】 山西医科大学 ， 劳动卫生与环境卫生学， 2012， 博士

【摘要】 第一部分亚慢性铝暴露对大鼠神经行为影响及其机制研究铝(aluminum, Al)是地壳中第三大化学元素,也是含量最多的金属元素,铝及其化合物在日常生活、生产中应用广泛,可通过饮水、食物、药物、烹调用具及粉尘等多种途径进入人体。铝是公认的强神经毒素,也是引起阿尔茨海默病(Alzheimer’s disease, AD)等多种神经退行性疾病的重要环境危险因素之一。铝在AD发生中的作用仍有争议,可能与实验用含铝化合物的种类及其在生理pH值条件下的化学形态有关。麦芽酚铝[aluminum-maltolate, Al(mal)3]是一种呈电中性的含铝复合物,在pH=7时能形成亚稳溶液,有利于进行铝神经毒性的相关研究。行为学改变是铝神经毒性的早期监测指标;海马长时程增强(long-term potentiation, LTP)足一个公认的与学习记忆有关的电生理模型；铝的过量接触和蓄积可损害学习记忆功能,导致老年痴呆样病症；神经系统病理改变是铝致认知功能损害的基础。本部分内容采用亚慢性腹腔注射Al(mal)3染毒SD大鼠建立神经系统损伤模型,从神经行为学、电生理、组织病理学及体内铝蓄积探讨铝的神经毒性。第一章亚慢性铝暴露对大鼠神经行为的影响目的：探讨Al(mal)3对大鼠神经系统的毒性作用。方法：采用Morris水迷宫(Morris water maze. MWM)、旷场实验(open field test. OFT)对2月龄健康雄性SD大鼠进行行：为学筛查,取学习记忆能力在5%-95%范围内的大鼠40只作为研究对象,按体重随机分为4组：生理盐水组、10μM/kg、20μM/kg、40μM/kg Al(mal)3染毒组。采用亚慢性腹腔注射Al(mal)3进行染毒,0.2ml/d,共60d。采用MWM、跳台实验(step-down test, SDT)、避暗实验(step-through test, STT)测定大鼠学习记忆能力；采用OFT测定大鼠自主活动能力。结果：亚慢性腹腔注射Al(mal)3染毒可导致大鼠MWM定位航行实验潜伏期相对延长,但差异无统计学意义(P>0.05);空间探索实验中,与生理盐水组比较,20μM/kg.40μM/kg Al(mal)3染毒组找到平台时间明显延长(P<0.05)；各Al(mal)3染毒组穿越原平台位置次数明显减少(P<0.05)。相关分析显示,铝暴露剂量与找到平台时间(rs=0.664,P<0.05),穿越平台位置次数(rs=-0.641,P<0.05)之间存在明显相关关系。SDT实验发现,Al(mal)3染毒组潜伏期明显缩短^,错误次数1和2明显增加,除低剂量Al(mal)3染毒组错误次数2外,其他各染毒组上述三指标与生理盐水组比较,均具有统计学意义(P<0.05)。染毒组大鼠STT潜伏期逐渐缩短,错误次数1和2逐渐增加,与生理盐水组相比,差异有统计学意义(P<0.05)。相关分析显示,铝暴露剂量与学习成绩(rs=0.789,P<0.01),潜伏期(rs=-0.708,P<0.01),记忆成绩(rs=0.466,P<0.05)之间存在明显相关关系。在OFT中,随染毒剂量的升高,各染毒组大鼠中央格停留时间明显延长,竖起次数、跨格次数明显减少,除低剂量染毒组竖起次数外,其余各染毒组上述观察指标与生理盐水组相比,均有统计学意义(P<0.05)。结论：亚慢性Al(mal)3染毒对大鼠神经系统具有明显损伤作用。第二章亚慢性铝暴露对大鼠LTP的影响目的：探讨亚慢性腹腔注射Al(mal)3对大鼠海马LTP的影响。方法：神经行为测试后,从每组随机取4只大鼠,采用电生理学方法进行海马LTP的测定。结果：与生理盐水组相比,Al(mal)3染毒组大鼠海马LTP受到明显抑制(P<0.05)。除10μM/kg Al(mal)3组在高频刺激后10min外,各Al(mal)3染毒组在高频刺激(high-frequency stimuli, HFS)后其余各时点与生理盐水组相比,标化的群体兴奋性突触后电位(field excitatory postsynaptic potential, fEPSP)的振幅明显降低(P<0.05)。结论：Al(mal)3可抑制大鼠海马CAl区LTP。第三章亚慢性铝暴露对大鼠海马形态结构的影响目的：探讨亚慢性腹腔注射Al(mal)3对大鼠海马形态结构的影响。方法：采用HE染色、普通光镜观察海马的大体结构,采用透射电镜观察海马的超微结构。结果：光镜观察发现,生理盐水组海马区神经细胞排列整齐,随着染铝剂的增高,细胞排列逐渐变凌乱,高剂量组可见细胞连接松懈,细胞核固缩,细胞周围出现环状带。电镜下可见生理盐水组细胞核形态规则,染色质分布匀匀,线粒体形态规则,嵴排列整齐,而随着染铝剂量的增高,细胞核皱缩,染色质边聚,核膜破裂,线粒体逐渐出现肿胀,破裂并可伴有线粒体嵴减少或消失。结论：亚慢性Al(mal)3染毒可导故大鼠海马结构严重受损。第四章亚慢性铝暴露对大鼠血铝、脑铝含量的影响目的：探讨亚慢慢性腹腔注射Al(mal)3对大鼠脑铝、血铝含量的影响。方法：动物分组及染毒同第一章,LTP测试结束后,采用石墨炉原子吸收分光光度法(graphite furnace atomic absorption spectrometry)测定大脑皮质、血清中铝含量。结果：脑铝含量均随染毒剂量的升高而增高,与生理盐水组比较有统计学差异(P<0.05)。结论：亚慢性Al(mal)3染毒可导致大鼠体内铝蓄积。第二部分铝暴露对淀粉样蛋白生成的影响AD是以进行性记忆减退、认和障碍和人格改变为主要临床表现的神经退行性疾病,老年斑(senile plaques, SPs)、神经纤维缠结(Neurofibrillary tangles. NFTs)和神经元丢失是其特征性病理表现。β-淀粉样蛋白(β-amyloid protein, Aβ)是构成SPs的主要成分,也是NFTs及血.管淀粉样变性的生化基础。Ap是种由40-43个氨基酸所构成的难溶性蛋白质,由淀粉样蛋白前体(amyloid protein precursor. APP)经淀粉样裂解途径所产生。Aβ的过度产生和蓄积可能是各种原因诱发AD的共同途径,是AD形成和发展的关键因素。长期摄入美国居民日常膳食规定铝量可导致大鼠海马和大脑皮层的APP过度表达,引起Aβ级联反应;大鼠神经元细胞暴露于亚致死浓度的三氯化铝(50μM)3周以上,可出现Aβ免疫阳性沉淀；铝可促进Aβ由α-螺旋向β-片层结构转变,加速Aβ生成和聚集,增加Aβ低聚物的稳定性。有研究认为铝可能通过直接影响Aβ合成代谢而导致实验动物脑内Aβ含量增加,但其确切机制目前尚不清楚。解聚素和金属蛋白酶-10(Adisintegrin and metalloproteinase-10, ADAM10)是最主要的α-分泌酶。APP的β-位点裂解酶1(β-site AβPP cleaving enzyme, BACE1)被认为是脑中涉及到Aβ产生的主要β-分泌酶。早老素1(presenilin1,PS1)是γ-分泌酶的重要催化成份。γ-分泌酶对APP的裂解能够产生Aβ40和Aβ42。Aβ42更易水解和形成原纤维,是AD老年斑的主要成份。本部分将采用动物实验、体外实验,分别对APP淀粉样物质代谢途径和非淀粉样物质代谢途径中的APP、ADAM10/17/9、BACE1和PSl的基因、蛋白表达,BACE1活性及Aβ40和Aβ42含量进行测定,探讨铝对APP代谢的影响。第一章亚慢性铝暴露对大鼠淀粉样蛋白生成的影响目的：探讨Al(mal)3对大鼠APP分解代谢的影响。方法：动物分组及染毒同第一部分,分别采用ELISA、qRT-PCR测定APP、ADAM10、ADAM17、ADAM9、BACE1和PSI的基因、蛋自表达,采用ELISA法测定BACE1酶活性及Aβ40和Aβ42含量进行测定。结果：随Al(mal)3暴露剂量的升高,大鼠皮质APP蛋白、基因表达逐渐增加,高剂量组APP蛋白、基因表达与生理盐水组比较,具有统计学差异(P<0.05)。高剂量Al(mal)3染毒组ADAM10、ADAM17和ADAM9的蛋白和基因表达较生理盐水组明显降低,差异具有统计学意意义(P<0.05)。中、高剂量Al(mal)3染毒组BACEl基因表达和蛋白含量与生理盐水组比较,均具有统计学差异(P<0.05)。中、高剂量。Al(mal)3染毒组BACEl酶活性与生理盐水组比较明显升高,具有统计学意义(P<0.05)。PSI蛋白表达随Al(mal)3染毒剂量的升高而逐渐增加,各Al(mal)3染毒组PSI蛋白含量与生理盐水组比较均具有统计学意义(P<0.05)。随染毒剂量的升高,Aβ40含量逐渐降低而Aβ42含量逐渐升高,中、高剂量Al(mal)3染毒组Aβ40含量与生理盐水组比较具有统计学差异(P<0.05)。低、中、高剂量Al(mal)3染毒组Aβ42含量与生理盐水组比较具有统计学意义(P<0.05),结论：铝可通过增强APP的淀粉样水解,抑制其非淀粉样水解而导致Aβ42生成增多。第二章铝暴露对PCl2细胞淀粉样蛋白生成的影响目的：控讨Al(mal)3染毒对PCl2细胞APP分解代谢的影响。方法：PC12细胞在Al(mal)3终浓度为0,50,100,200,400pM的培养基中培养12h,24h,48h后,采用ELISA法测定APP、ADAM10、ADAM17、ADAM9、BACE1、PS1、Aβ40和Aβ42蛋白含量,采用qRT-PCR测定APP、ADAM10、ADAM17、ADAM9、BACE1、PS1基因表达,采用ELISA法测定BACE1酶活性。结果：400μM Al(mal)3染毒PC12细胞12h,24h,48h后,其APP蛋白、基因表达明显增加,与对照组比较具有统计学差异(P<0.05)。50,100,200,400μM Al(mal)3染毒PC12细胞12h,24h,48h后,ADAM10蛋白和基因表达均显著下降,与对照组比较,差异具有统计学意义(P<0.05)。50,100,200,400μM Al(mal)3染毒PC12细胞24h,48h后,ADAM17和ADAM9蛋白和基因表达均显著下降,与对照组比较,差异具有统计学意义(P<0.05)。Al(mal)3染毒可导致PC12细胞BACE1蛋白、基因表达增加。400μM A1(mal)3染毒PC12细胞12h,24h,48h后,其BACE1蛋白和基因表达与对照组比较明显增加(P<0.05)。随着染毒剂量的增加和染毒时间的延长,PC12细胞Aβ40表达逐渐减少而Aβ42表达逐渐增加。结论：体外实验证实,Al(mal)3对APP的淀粉样水解具有明显促进作用,同时对分淀粉样水解具有明显抑制作用。第三部分铝暴露对Reelin信号转导通路的影响脑中Aβ的过度表达是导致AD发病的主要因素。Aβ是由APP经β-,γ-分泌酶连续水解所形成。一些研究认为,APP和β-分泌酶均存在与细胞表面,被内吞进入内涵体后产生大量Aβ,相关分子机制尚不清楚。Reelin是一种大型的细胞外糖蛋白,Reelin可与细胞膜表面受体ApoER2和VLDLR受体结合,通过胞内衔接蛋白Dab1将Reelin信号转导入细胞内。Reelin信号可引起Dab1依赖的涉及多种激酶的信号级联反应。在神经系统发育期间,可以调节神经元的正确迁移和定位。而且,Reelin信号通路被认为参与了神经退行性疾病的发生。近年来,越来越多的证据表明,Reelin及其信号通路的组分与AD特征病理表现相关。AD转基因小鼠动物模型研究发现Reelin存在于老年斑中。Dab1可增加转染细胞和原代培养细胞膜表面APP农达,增加APP被α-分泌酶裂解,减少Aβ生成。Reelin可影响Dab1的这种作用。Reelin能够显著增加Dab1和APP的免疫共沉淀,而且,Reelin可减少Aβ产生。因此,Reelin及其信号转导通路成分可能参与了APP的运输及裂解过程。但确切制目前尚不清楚。本部分采用体内和体外实验,探讨Al(mal)3致APP代谢异常过程中Reelin信号转导通路相关成分的表达变化。第一章亚慢性铝暴露对大鼠Reelin信号转导通路相关成分蛋白、基因表达的影响目的：探讨铝暴露对大鼠皮质Reelin及其信号转导通路相关基因表达的影响。方法：动物分组及染毒同第一章,采用ELISA法测定大鼠大脑皮质Reelin、ApoER2、VLDLR、 Dab1蛋白含量,采用qRT-PCR测定大鼠大脑皮质Reelin基因(RELN).ApoER2、VLDLR、 Dab1基因表达。结果：随染毒剂量升高,Reelin蛋白及基因表达逐渐下降,各剂量Al(mal)3染毒组大鼠皮质Reelin蛋白含量及RELN基因表达与对照组比较均具有统计学差异(P<0.05)。中、高剂量Al(mal)3染毒组VLDLR基因农达较对照组明显升高而蛋白含量却显著下降,与对照组比较,差异具有统计学意义(P<0.05)。各Al(mal)3染毒组大鼠皮质ApoER2基因表达较对照组显著下降(P<0.05),高剂量Al(mal)3染毒组大鼠皮质ApoER2蛋白含量较对照组明显降低,差异具有统计学意义(P<0.05)。各染毒组Dab1基因表达与对照组比较明显下降,差异具有统计学意义(P<0.05),中、高剂量组Dab1蛋白含量较对照组明显降低(P<0.05)。结论：亚慢性铝暴露可影响大鼠皮质Reelin信号转导通路相关成分的基因和蛋白表达,铝致Aβ合成异常可能与此有关。第二章铝暴露对PC12细胞Reelin信号转导通路相关成分蛋白、基因表达的影响目的：探讨铝暴露对PC12细胞Reelin及其信号转导通路相关基因、蛋白表达的影响。方法：细胞染毒同第二部分,采用ELISA法测定PC12细胞Reelin、ApoER2、VLDLR、 Dab1蛋白含量,采用qRT-PCR测定PC12细胞RELN、ApoER2、VLDLR、Dab1基因表达。结果：Reelin蛋白和基因表达随染毒剂量升高而逐渐减少,200、400μM Al(mal)3染毒PC12细胞12h,24h和48h,Reelin蛋白和基因表达较对照组明显降低(P<0.05)。200、400μMAI(mal)3染毒组VLDLR蛋白和基因表达较对照组明显下降(P<0.05)。除50μMAl(mal)3染毒PC12细胞24h外,其余各染毒组PC12细胞染毒12h,24h和48h均可导致ApoER2蛋白和基因表达明显下降(P<0.05)。各剂量AI(mal)3染毒PC12细胞24h和48h均可导致Dab1蛋白和基因表达显著下降,与对照组比较具有统计学差异(P<0.05)。结论：铝暴露可影响PC12细胞Reelin信号转导通路相关成分的蛋白和基囚的表达。结论：1.铝致Aβ生成增多在铝致学习记忆障碍和AD等神经退行性疾病发生过程中起十分重要的作用。铝致Aβ生成增多的主要机制为：促进BACE1和PSI表达,增加BACE1活性,使APP水解以淀粉样物质途径为主,同时降低ADAM10/17/9表达,进一步减少APP的非淀粉样水解途径：2. Reelin信号转导通路可能参与了铝致APP水解异常、Aβ生成增多过程的调节。更多还原

【Abstract】 Part I Study on the effects of sub-chronic exposure to aluminum on neurobehavior and its mechanism in ratsAlinninum(Al), the most abundant metal and the third largest chemical element in the earth crust,is widely used in our day-to-day life and in the course of industrial production and gains an easy access to our body through using of drinking water, foods, cooking utensils, some antacids, Al-containing dusts and fumes. Al is a potent neurotoxicant and has been proposed as one of the critical environmental factors in several neurodegenerative diseases such as Alzheimer’s disease (AD). The role of Al in AD remains controversial, which may be related with the chemical species of Al and the chemical form of the metal ion at neutral pH. Aluminum-mallolate complex [Al(mal)3] is a electroneutral complex of Al. which forms a metastable solution and can deliver a significant amount of free aqueous Al at physiological pH. Behavioural changes may be an monitoring index of neurotoxicity and may be observed early during the exposure to Al. Long term potentiation(LTP) is widely used as a well-known model to investigate the cellular mechanisms underlying some kinds of learning and memory. Al accumulation in both the brain and serum is assumed to impair learning and memory, lead to dementia-like symptoms. Changes in the structure of the nervous system is the basis of Al neurotoxicity. In the section, neurological damage model was established by sub-chronic intraperitoneal injections of Al(mal)3in rats. The neurotoxicity of Al was evaluated by neuroethology, electrophysiology. histopathology and the level of Al in both the brain and serum.Chapter I Effect of sub-chronic exposure to aluminum on neurobehavior in ratsObjective To expose the neurotoxicity of Al(mal)3. Methods Behavior screening on two months old Sprague Dawley(SD) male rats was carried out by Morris water maze(MWM) and open-field test(OFT). Thirty two male SD rats were divided randomly into four subgroups according to the body weight:saline group,10,20. and40μM/kg Al(mal)3treated groups. Over two months, rats in the saline group received daily intraperitoneal (i.p.) injections0.9%saline, rats in the10,20, and40μM/kg Al(mal)3groups received i.p. administrations of these three doses, respectively. After exposure, the MWM, step-down test(SDT), step-through test(STT) were performed to examine the learning and memory abilities of rats, the OFT was performed to exmine the general activities of rats. Subsequently, LTP in hippocampus was measured. Al concentration in both cerebral cortex and blood were determined by graphite furnace atomic absorption spectrometry. The morphological changes of hippocampus were observed by optical microscope and electron microscopy. Results In the same day, escape latencies in the learning acquisition test of Al(mal)3groups were relatively prolonged when compared to those of the saline group, but there was no statistically significant difference (P>0.05). In the probe trail, the time of finding platform site of20,40μM/kg Al(mal)3groups increased progressively compared to those of saline group (P <0.05), the average number of crossings of the platform site was significantly reduced in the Al(mal)3group compared to those of saline group (P<0.05). There were significantly correlationship between the dose of Al exposure and the time of finding the platform site (r,=0.664, P<0.05), Al exposure dose and the average number of crossings of the platform site (rs=-0.64l. P<0.05). In the SDT. the significant decrease was found in the mean latency time of all Al(mal)3groups when compared with those of saline group (P<0.05). while errorcountland2were increased significantly in Al(mal)3groups, there were obvious significance in all Al(malb groups, except errorcount2in10uM/kg Al(mal)3groups, compared to the saline group (P<0).05). There were correlationship between the dose of Al exposure and errorcountl of SDT (rs=0.663. P<0.01). Al exposure dose and errorcount2of SDT (rs=0.943. P <0.01). The latency time of SIT in Al(mal)3exposed rats decreased gradually, while the errorcount1and2was increased significantly along with the increase of Al dosage. There were obvious significance in all Al(mal)3groups when compared to the saline group (P<0.05). Statistical analysis confirms that a correlationships between the dose of Al exposure and errorcount1(rs=0.789. P<0.01), the dose of Al exposure and latent period of STT (rs=-0.708. P<0.01), furthermore, the dose of Al exposure and errorcount2(rs=0.466, P<0.05). The retention time in the centre of the arena was significantly longer, while The total number of squares crossed and the total numbers of rearings in OFT were decreased significantly along with the increase of Al dosage, except the total numbers of rearings in10μM/kg Al(mal)3groups (P <0.05). Conclution Sub-chronic exposure to Al(mal)3has a significant injury of the nervous system in rats.Chapter Ⅱ Effect of sub-exposure to aluminum on LTP in hippocampal CA1area in ratsObjective To investigate the effects of Al(mal)3on the hippoeampal Long-term potentiation (LTP) in vivo. Methods Animals and treatments were same with chapter I. After the behavioral investigations, four rats were randomly elected from every group, LTP of the Schaffer collateral-CA1pathway was recorded. Results Compared with saline group, a significant suppression of LTP by Al(mal)3was found. Although the average standardized fEPSP amplitude at the beginning did not obviously change, the values gradually decayed towards the baseline during1h of recording. At10min,20min,30min,40min,50min and60min after HFS, the average standardized fEPSP amplitude in all of Al(mal)3groups, except10μM/kg Al(mal)3group at10min after HFS, were significantly decreased compared to those in saline group (P<0.05). Conclution The result indicates that the Al(mal)3induced impairment of LTP in hippocamcal CA1of rats.Chapter III Effect of sub-exposure to aluminum on morphological structure of hippocampus in ratsObjective To investigate the effects of Al(mal)3on morphological structure of hippocampus in rats. Methods Animals and treatments were same with chapter Ⅰ. Morphological structure of hippocampus was observed with light microscope and electron microscope. Results The observation by optical microscope showed:arrangement of the neurocytes in rats’hippocampus was orderly in control group, but with the increasing of the doses of Al exposure, the neurocytes displayed disorderly, the connections among the neurocytes were loosened, the karyon shrinked, some rings around the neurocyte were observed. Picture by electron microscope displayed:the karyotin distributed symmetrically, and its form was regular, the karyon was unbroken, and with the increasing of the doses of Al exposure, the karyon shrinked, the karyotin assembled in the edge, karyotheca was broken. Conclution The result indicates that the Al(mal)3can cause obviously serious damage of hippocampal formation.Chapter Ⅳ Effect of sub-exposure to aluminum on Al contents in both cerebral cortex and blood in ratsObjective To investigate the effects of Al(mal)3on Al contents in both cerebral cortex and blood. Methods Animals and treatments were same with chapter Ⅰ. After the LTP were recorded. Al contents in both cerebral cortex and blood were assayed by graphite furnace atomic spectrophotometry. Results Al contents in both cerebral cortex and blood in Al(mal)3exposed groups were increased significantly along with the increased of Al(mal)3exposure dosage, which was significantly higher than those in saline group(P<0.05). Conclution The result indicates that the Al(mal)3can cause obviously Al accumulation.Part II Effect of exposure to aluminum on the production of amyloid protein in vitro and in vivo AD, a devastating neurodegenerative disease, which is clinically characterized by the progressive memory loss, cognitive impairment, personality changes, and is pathologically characterized by senile plaques (SPs), intraneuronal neurofibrillary tangles (NFTs), and severe neuronal loss. Aβ, a39to43amino acid peptide, is not only a major component of senile plaque but also the biochemical basis of NFTs and vascular amyloidosis. A(3, generated by proteolytic cleavage of the amyloid precursor protein(APP), plays a pivotal role in the pathogenesis of Alzheimer’s disease (AD). the excessive producation and accumulation of Aβ may be the common way to AD induced by various reasons. Walton et al. founded that the gene expression of APP in the hippocampal and cortical tissue was elevated, when rats were chronically ingested Al in amounts equivalent to total dietary Al levels that Americans routinely ingest. Al may thus launch the cascade that results in the formation of amyloid plaques. It was reported that long-term exposure to sublethal levels of aluminium chloride (50μM) for more than3weeks caused depositions immunopositive to Aβ in cultured neurons. Al can promote the structural changes of Aβ from a-helix to β-sheet, accelerate Aβ generation and aggregation, increase the stability of Aβ oligomers. Some studies suggested that Al may cause Aβ increased in experimental animals by promoting directly the synthesis of Aβ. but its exact mechanism is unclear. A disintegrin and metalloproteinase10(ADAM10) is the constitutive α-sccretase.β-site AβPP cleaving enzyme (BACE1) is thought to be the major β-secretase involved in Aβ production in the brain.The BACE1enzyme is essential for the generation of Aβ. BACE1cleavage of APP is a prerequisite for Aβ formation and is putatively the rate-limiting step in Aβ genesis. Presenilin1(PSI) are the crucial catalytic components of y-secretase.γ-cleavage can yield both AP40and AP42. AP42is more hydrophobic and more prone to fibril formation than others, and is also the predominant isoform found in cerebral plaques in AD. In this section, we will test the protein and gene expression of APP. ADAM10, ADAM17, ADAM9, BACE1and PS1in brain in rats and in PC12cell, respectively. At the same time, BACE1activity and Aβ40and Aβ42content was determined to explore the effects of Al on the metabolism of APP.Chapter I Effect of sub-chronic exposure to aluminum on the production of amyloid protein in ratsObjective To expose the effect of Al(mal)3on the catabolism of amyloid precursor protein in rats. Methods Animal and treatment were same with those of part Ⅰ. After exposure, the protein and mRNA expression of ADAM10. ADAM17, ADAM9. BACE1, PS1were detected by quantitative PCR and ELISA in cerebral cortex. The BACE1activity were detected by β-secretase activity assay kit. The protein expression of Aβ40and Aβ42in cerebral cortex were detected by ELISA. Results The protein and mRNA expression of APP were increased gradually with increasing Al(mal)3dosage. The protein and mRNA expression of APP in40μM/kg AI(mal)3-exposed group were significantly higher than those of the saline group (P<0.05). The protein and mRNA expression of ADAM10, ADAM17, ADAM9in40μM/kg Al(mal)3-exposed group were significantly higher than those of the saline group (P<0.05). The protein expression of BACE1in20,40μM/kg Al(mal)3-exposed groups were significantly increased when compared to those of the saline group (P<0.05). The mRNA expression of BACE1in20,40μM/kg Al(mal)3-exposed group was significantly higher than those of the saline group (P<0.05). When compared with the saline group, the enzyme activity of BACE1it20,40μM/kg Al(mal)3-exposed groups were significantly increased(P<0.05). The protein expression of PS1were increased gradually with increasing Al(mal)3dosage. The protein expression of PS1in all Al(mal)3-exposed group were increased significantly than those in saline group(P<0.05). The mRNA expression of PS1in40μM/kg Al(mal)3-exposed group was significantly higher than those of the saline group (P<0.05). The protein expression of Aβ40were decreased gradually and the protein expression of Aβ42were increased gradually with increasing Al(mal)3dosage. The protein expression of Aβ40in20,40μM/kg Al(mal)3-exposed groups were significantly decreased when compared to those of the saline group (P<0.05). The protein expression of Aβ42in all Al(mal)3-exposed group were significantly increased when compared to those of the saline group (P<0.05). Conclution Aluminum can lead to an increase in the protein expression of Aβ42by enhancing the amyloid hydrolysis of APP and inhibiting the non-amyloid hydrolysis of APP in rats.Chapter II Effect of exposure to aluminum on the production of amyloid protein in PC12cellsObjective To expose the effect of Al(mal)3on the catabolism of amyloid precursor protein in PC12cells. Methods Rat pheochromocytoma cell line(PCl2cells) were incubated in culture media with Al(mal)3at final concentration of0,50,100,200,400μM for12h.24h. and48h, respectively. The protein expression of APP, ADAM10, ADAM17, ADAM9, BACE1, PS1, A(340, and A(342were determined by ELISA. The mRNA expression of APP. ADAM10, ADAM17. ADAM9, BACEI, PS1were detected by quantitative PCR. The enzymatic activity of BACE1were determined by ELISA. Results Treatment of PC12cells with200μM.400μM Al(mal)3for12h,24h and48h did show a significant increase in the protein and mRNA expression of APP except the protein expression of APP at200μM/kg AI(mal)3group for24h (P<0.05). Al(mal)3can increase the protein and mRNA expression of BACE I in PC12cells. When the PC12cells were treated with Al(mal)3for12h.24h and48h. the protein and mRNA expression of ADAM10in50,100,200,400μM Al(mal)3groups were significantly decreased when compared to those of control groups(P<0.05). The protein and mRNA expression of ADAM17, ADAM9in PC12cells were treated with400μM Al(mal)3for24h and48h were significantly decreased when compared to those of control groups(P<0.05). The protein and mRNA expression of BACE1in PC12cells were treated with400μM Al(mal)3for12h,24h and48h were significantly increased when compared to those of control groups(P<0.05). The protein expression of Aβ40in PC12cells were decreased gradually and the protein expression of Aβ42in PC12cells were increased significantly along with the increase of Al(mal)3dosage and exposed time(P<0.05). Conclution Results in vitro experiment indicated that Al(mal)3can accelerate the amyloidogenic processing of APP, at the same time, inhibit the non-amyloidogenic processing of APP.Part III Effect of exposure to aluminum on Reelin-ApoER2/VLDLR-Dabl in RatsThe excess expression of Aβ in brain is a major factor in the pathogenesis of Alzheimer’s disease (AD). A(3is produced through a sequential cleavage of APP by (3-secretase and y-secretase. Some studies suggested that APP and β-secretase, both present on cell surface, are endocytosed into endosomes to produce Aβ. The molecular mechanism by which neurons trigger the production of Aβ is poorly understood.Reelin is an extracellular glycoprotein that binds to the transmembrane receptors apolipoprotein-E receptor type2(ApoER2) and very low density lipoprotein receptor (VLDLR). which transduce the Reelin signal through the intracellular adapter Disabled-1(Dabl). Reelin signaling triggers a Dab-1-dependent signaling cascade involving several kinases, which ultimately controls proper neuronal migration and positioning during CNS development. Furthermore, the involvement of the Reelin signaling pathway in neurodegeneration has also been proposed. In recent years, increasing evidence supports a link between Reelin and components of its signaling pathway with the main hallmarks of AD pathology. Recent study indicate that Reelin is present in β-amyloid plaques in a transgenic mouse model of AD. Dabl increases cell surface expression of APP. increases α-cleavage of APP, and decreases Ap in transfected cells and in primary neurons, and this effect is influenced by Reelin. Reelin treatment significantly increases co-immunoprccipitation of Dabl with APP. Moreover, Reelin decreases production of the Aβ. Therefore. Reelin and components of its signaling pathway may participate in APP trafficking and processing. The certain mechanism is largely unknown. In this part, the expression of Reelin and components of its signaling pathway were detected by qRT-PCR. when APP abnormal metabolic process were induced by Al (mal)3in vivo and in vitro. Chapter Ⅰ Effect of sub-chronic aluminum exposure on the expression of Reelin and components of its signaling pathway in ratsObjectsive To expose the effect of sub-chronic exposure to Al(mal)3on the gene expression of Reelin and components of its signaling pathway in rats. Methods Animal and treatment were same with Part Ⅰ. Results The mRNA and protein expression of Reelin decreased gradually with increasing of Al(mal)3doseage. The mRNA and protein expression of RELN in10,20,40μM Al(mal)3groups had statistically significant when compared with those of control groups(P<0.05). The mRNA expression of VLDLR increased significantly but the protein expression of VLDLR decreased significantly in20,40μM Al(mal)3groups when compared with those of control groups(P<0.05). The mRNA expression of ApoER2in all Al(mal)3groups decreased significantly when compared with those of saline groups(P<0.05). The protein expression of ApoER2in40μM Al(mal)3groups decreased significantly when compared with those of saline groups(P<0.05). The mRNA expression of Dabl in all Al(mal)3groups decreased significantly when compared with those of control groups(P<0.05). the protein expression of Dabl decreased significantly in20.40μM Al(mal)3groups when compared with those of control groups (P<0.05). Conclution Sub-chronic exposure to Al(mal)3had a significant effect on the protein and mRNA expression of Reelin and components of its signaling pathway in rats, which may be related to the abnormal composition of Aβ induced by Sub-chronic exposure to Al(mal)3.Chapter Ⅱ Effect of aluminum exposure on the expression of Reelin and components of its signaling pathway in PC12cellsObjectsive To expose the effect of Al(mal)3exposure on the protein and gene expression of Reelin and components of its signaling pathway in PC12cells. Methods PC12cells were incubated in culture media with Al(mal)3at final concentration of0,50,100,200,400μM for12h,24h, and48h, respectively. The protein and gene expression of Reelin, VLDLR, ApoER2, and Dab1were detected by ELISA and qRT-PCR, respectively. Results The protein and gene expression of Reelin decreased gradually with increasing Al(mal)3dosage. The protein and gene expression of Reelin in PC12cells which exposured to200,400μM Al(mal)3for12h,24h. and48h increased significantly when compared with those of control groups(P<0.05). The protein and gene expression of VLDLR in PC12cells treated with200,400μM Al(mal)3for12h,24h, and48h were decreased obviously(P<0.05). Treatment of PC12cells with50,100.200.400μM Al(mal)3for12h,24h and48h did show a significant decrease in the protein and mRNA expression of ApoER2(P<0.05). except the protein expression of ApoER2at50μM/kg AI(mal)3for24h (P>0.05). The mRNA and protein expression of Dabl in PC12cells treated with50,100,200,400μM Al(mal)3for24h,48h were decreased significantly when compared with those of control groups(P<0.05). Conclution The exposure to Al(mal)3had a significant effect on the protein and gene expression of Reelin and components of its signaling pathway in PC12cells.Conclusions:1. The excessive production of Aβ induced by sub-chronic exposure to aluminum may be play a major role in aluminum-induced learning and memory disorder, and several neurodegenerative diseases, particularly AD. Its main mechanism is Al accelerates amyloidiosis pathway of APP through promoting the expression of BACE1and PS1, at same time, Al inhibits nonamyloidosis pathway through reducing the expression of ADAM10, ADAM17, and ADAM9.2. Reelin signal transduction pathways may be involved in the adjustment of the abnomal hydrolysis of APP and the excessive production of Aβ induced by Al (mal)3.更多还原