【作者】 程淑群；

【导师】 王应雄；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2009， 博士

【摘要】 苯并[a]芘(B[a]P)广泛存在于生产和生活环境中,主要来源于有机物的高温裂解过程,是当前危害全人类健康最主要的化学污染物之一。以往对其毒性研究主要集中在致癌性方面,但其化学结构与理化特性提示其具有神经毒性。动物实验表明B[a]P可引起大鼠脑组织器质性损伤和行为学改变,人群流行病学研究也表明B[a]P为代表的PAHs具有肯定的神经毒性,其中最为突出的是影响暴露者的学习记忆能力,但目前有关B[a]P的神经毒性研究零散而不深入。本研究拟通过慢性动物实验,利用行为学、生理与生化、基因和蛋白芯片技术等方法,系统研究B[a]P对学习记忆能力的影响及影响机制。本研究共四部分。第一部分苯并[a]芘对大鼠海马形态学和学习记忆能力的影响初断乳雄性Wistar大鼠120只,根据Morris水迷宫测试结果分为5组:空白对照组、溶剂对照组、B[a]P高浓度组、中浓度组和低浓度组(分别为6.25、2.5和1mg/kg体重)。每周5次定时腹腔注射,连续13周(90天)。在染毒过程中,各组动物饮食、活动正常,无异常行为学改变,无动物死亡,成功建立了B[a]P慢性暴露的动物模型,为探讨B[a]P对大鼠学习记忆能力的影响,在染毒结束后,用Morris水迷宫对大鼠空间学习记忆功能进行评价。结果显示:随着暴露剂量的增加,各组大鼠寻找平台的平均逃避潜伏期延长,末次跨台次数明显减少,在目标区域游泳时间所占比例减少。各剂量组与对照组比较有显著性差异,高、中剂量组同低剂量组间差异有显著性(P＜0.05)。病理结果显示:HE染色对照组和B[a]P暴露各组大鼠海马神经元形态结构未见明显异常,神经元细胞排列整齐,核仁明显,神经纤维排列整齐。尼氏体染色各组海马神经元胞质呈均一蓝色,中、高剂量组见少许尼氏体溶解现象。电镜结果显示对照组和低、中剂量组海马神经元超微结构未见异常,高剂量组偶有线粒体肿胀和核浓缩现象出现。本研究结果显示较低剂量B[a]P慢性暴露可损害大鼠的空间学习记忆能力,但对海马神经元形态和超微结构可能无明显影响。第二部分苯并[a]芘对大鼠海马神经生物学的影响行为学测试结束后,大鼠断头处死,取出海马组织。应用相关试剂盒分别测定海马中一氧化氮(NO)、一氧化氮合酶(NOs)、超氧化物歧化酶(SOD)、丙二醛(MDA)、乙酰胆碱转移酶(ChAT)、胆碱酯酶(AChE);碱性羟胺法测定乙酰胆碱(Ach)含量;电化学高效液相色谱法测定去甲肾上腺素(NE)、肾上腺素(A)、3-4二羟基苯乙酸(DOPAC)、多巴胺(DA)、5-羟吲哚酸(5-HIAA)、5羟色胺(5-HT);免疫组化法测定海马谷氨酸(Glu)和γ-氨基丁酸(GABA)阳性表达。用透射电镜观察海马CA1区神经元突触形态。结果显示:B[a]P暴露高剂量组神经元突触PSD厚度和突触活性区长度均明显小于对照组和低剂量组(P＜0.05),突触界面半径明显大于对照组和低剂量组(P＜0.05);随着B[a]P暴露剂量的增加,SOD活性下降,MDA含量增加,各剂量与对照组间比较,差别有显著性意义(P＜0.05),各剂量组间比较,高剂量组与低剂量组间有显著性差异(P＜0.05);B[a]P暴露各组大鼠海马NOs活性和NO含量均低于对照组(P＜0.05),且随着B[a]P暴露剂量的升高,NOs活性和NO含量逐渐降低;与对照组比较,B[a]P暴露组大鼠海马ChAT活性降低、AChE活性升高、Ach含量降低(P＜0.05);B[a]P暴露组NE、DA、DOPAC和5-HT都较对照组显著增加(P＜0.05),随着B[a]P剂量的增加各指标有增加的趋势,且各剂量组间差别有显著性意义(P＜0.05)。本研究表明B[a]P慢性暴露可影响海马神经递质的含量,氨基酸神经递质降低和单胺类神经递质增高使LTP的诱导和维持受到抑制,影响学习记忆能力。NO/NOs和ACh含量降低、脂质氧化性损伤等均可能影响学习记忆能力。第三部分苯并[a]芘对大鼠海马整体基因表达的影响研究本部分首次利用基因芯片技术分析B[a]P对大鼠海马整体基因表达谱改变的影响,用定量PCR技术对筛选出来的基因进行验证。芯片结果显示:暴露组与对照组比较表达上调≥2倍的基因有440个,表达下调≥2倍的基因有582个,这些差异表达的基因涉及细胞周期调节因子、应激反应蛋白、离子通道及转运蛋白、细胞内信号传导调节因子、凋亡蛋白、DNA合成蛋白、肿瘤抑制基因、神经递质受体、细胞表面抗原、细胞间通信蛋白、细胞骨架与运动蛋白、转录因子及DNA连接蛋白、原癌基因等14大类功能相关基因。其中可能与学习记忆分子机制相关的基因包括:离子通道和神经递质传递相关基因(Kcnh4、Kcnab3、Kcnn4、Syt2、Synpr下调;Htr1a、Htr5b、Gabra5、Nrxn1、Calb3上调);信号传导相关基因(Prkcd、Prkch、Snf1lk、Ppm1f、Rab27a、Htr1d、PSD、G naz下调;Trpc4、Mapk2上调);转录相关基因(c-Fos、Nr2f6、Egr2下调;Erg、BNDF上调);结构/细胞骨架相关基因(Myl1、Mybpc、Gcnt2、MyoⅠc下调;Myh4上调)。结果表明在B[a]P暴露下,学习记忆能力下降涉及多基因协同调控,主要是离子通道和神经递质传递、信号传导、转录、结构/细胞骨架等相关基因的表达下调或上调。第四部分苯并[a]芘对大鼠海马整体蛋白表达的影响研究近年来,海马蛋白质组学越来越多地应用于学习记忆机制的研究。本部分利用Springbio高通量抗体芯片分析对照组和B[a]P暴露组大鼠海马蛋白表达变化,从蛋白质组学角度探讨B[a]P影响学习记忆能力的机制。芯片结果显示:暴露组与对照组蛋白表达比较,暴露组26个蛋白质上调≥1.5倍,23个蛋白质下调≥1.5倍。其中可能参与学习记忆机制调节的蛋白如视黄酸受体b(RAR b)、脑源性神经营养因子(BDNF)和突触结合蛋白1(Sty1)分别下调2.6、2.1和1.6倍,提示B[a]P可能通过减少RAR b、BDNF、Sty1在海马内的表达而损害学习记忆能力。总结:较低剂量B[a]P慢性暴露可损害大鼠的空间学习记忆功能,可能与海马氨基酸神经递质、NO/NOs、Ach含量降低、单胺类神经递质增高以及脂质氧化性损伤等有关。B[a]P暴露损害学习记忆能力涉及多基因协同调控,也可能与RAR b、BDNF、Sty1等蛋白在海马内的下调有关。但B[a]P影响学习记忆能力的确切分子机制尚有待进一步研究。更多还原

【Abstract】 Benz[a]pyrene(B[a]P),a product of incomplete combustion at temperatures between 300℃and 600℃,widely existed in ecosystem,is one of key hazardous chemicals that can harm human health on the earth.A vast number of studies over the previous three decades have documented links between B[a]P and cancers.According to the chemical structure and physicochemical properties of B[a]P,scientists infer that it might be a neurotoxin.Animal studies showed that B[a]P induce impairment of brain and alterations in behavior,and epidemiology researches also found that B[a]P is a neurotoxin certainly,especially impacting on learning and memory behavior.Evidences that B[a]P cause neurotoxicity,however,are still not complete.The need for more research on neurotoxicity of B[a]P becomes increasingly important as the combustion of fossil fuel increases to keep pace with the world’s energy needs.Therefore the aim of this study is to acquire systemic knowledge about the effect of B[a]P on learning and memory behavior in rats and mechanisms involved in it.PARTⅠEFFECTS OF BENZ[A]PYRENE ON HIPPOCAMPUS MORPHOLOGY AND CAPABILITY OF LEARNING AND MEMEORY IN RATSAccording to the results of water maze test,one hundred twenty weaned male Wistar rats were divided into 5 groups,including a control group,a solvent control group and three B[a]P -treated groups,which were administered intra-peritoneally with three doses from 1.0,2.5 to 6.25 mg/kg body weight(BW) for 13 weeks(90 d).During 90 d,there is no change of rats’ behavior and no animal died.Model of rat exposed to Benz[a]pyrene for 90 d is established successfully.After 90 d,Morris water maze test is applied to assess the effects of B[a]P on capability of the learning and memory in rats.The test results showed,with the increasing of the dose exposed to B[a]P,that the average escape latency of rats increases,the frequency of crossing platform decreases and the time of swimming in the target area decreases.There are significant differences between the treated groups and the control groups and among three treat groups(P＜0.05).Results of pathological sections stained with hematoxylin and eosin showed that hippocampal neurons from all groups exhibited a typical normal appearance.Results of pathological sections stained with thionine for Nissl bodies showed that cytoplasm of hippocampal neuron was blue and Nissl bodies were very numerous in all groups,except slight chromatolysis appeared in 2.5 and 6.25 mg/kg B[a]P treat group.Electron micrographs confirmed the normal appearance of hippocampal neurons, except a few swelled mitochondrias appearing in 6.25 mg/kg treat group.The study of this part suggested that low dose of B[a]P for 90 d toxicological test could impair the capability of learning and memory in rats,but did not impair the morphology and structure of hippocampal neuron.PARTⅡEFFECTS OF BENZ[A]PYRENE ON HIPPOCAMPUS NEUROBIOLOGYAfter the Morris water maze test,hippocampus tissues were obtained from decapitated rats.Nitric oxide(NO),nitric oxide synthase(NOs), superoxide dismutase(SOD),malondialdehyde(MDA),choline cetyltransferase(CHAT),acetylcholine esterase(ACHE) were detected by commercially available kits.Acetylcholine(Ach) was detected by alkaline hydroxylamine method.Norepinephrine(NE),adrenaline(A), 3,4-dihydroxyphenylacetic acid(DOPAC),dopamine(DA), 5-hydroxyindoleacetic acid(5-HIAA),5-hydroxytryptamine(5-HT) were detected by high-performance liquid chromatography and electrochemical detection.Gliutamic acid(Glu) andγ-aminobutyric acid(GABA) were detected by immunohistochemistry.CA1 hippocampal synapse morphology was observed by transmission electron microscopy.Results of transmission electron microscopy showed that length of postsynaptic dlensity(PSD) and length of active zone were significant shorter than those of control groups(P＜0.05),and radius of modeling circular of synaptic interface was significant larger than those of controls(P＜0.05). With the increasing of the dose exposed to B[a]P,activity of SOD decreasing,concentration of MDA increasing,there were significant differences between the treat groups and the control groups and among three treat groups(P＜0.05).Activity of NOs and concentration of NO in treat groups were lower than those of control groups(P＜0.05),and were a decreasing trend companied with the increasing of the dose.Compared with control groups,activity of ChAT decreased,activity of AChE increased,concentration of Ach decreased(P＜0.05).Concentration of NE, DA,DOPAC and 5-HT in treat groups were lower than those of control groups(P＜0.05),and there were an increasing trend companied with the increasing of the dose and significant differences among three treat groups (P＜0.05).The study of this part suggested that low dose of B[a]P for 90 d toxicological test could alter the concentration of neurotransmitter to inhibit the inducement and sustainment of long-term potentiation(LTP), further leading to impair the capability of learning and memory in rats. The decreasing concentration of NO/NOs and Ach,and impairment of lipid oxidation could be related to effect the capability of learning and memory.PARTⅢEFFECTS OF BENZ[A]PYRENE ON WHOLE HIPPOCAMPUS GENE EXPRESSIONOur study original applied microarray technique to analyze the whole hippocampus gene expression exposed to B[a]P,then confirmed a part of results of microarray by RT-qPCR.The results of microarray showed that 440 genes were up-regulated by≥2 folds and 582 genes were down-regulated by≥2 folds,when treat group compared to control. Function genes were involving in 14 types,including in cell cycle regulatory factors,stress response proteins,ion channel and transporters, intracellular signal transduction regulatory factors,apoptosis proteins, DNA synthesis protein,tumor suppressor gene,neurotransmitter receptors, cell surface antigens,intercellular communication protein,cytoskeleton and movement protein,transcription factor,DNA connexin and proto-oncogene.Among these,genes perhaps associated with mechanisms of learning and memory involved:ion channel gene and neurotransmitter gene related to transmission(Kcnh4,Kcnab3,Kcnn4,Syt2,Synpr down-regulated;Htr1a,Htr5b,Gabra5,Nrxn1,Calb3 up-regulated);genes related to signal transduction(Prkcd,Prkch,Snf1lk,Ppm1f,Rab27a, Htr1d,PSD,G naz down-regulated;Trpc4,Mapk2 up-regulated);genes related to transcription(c-Fos,Nr2f6,Egr2 down-regulated;Erg,BNDF up-regulated);genes related to cell structure/cytoskeleton(Myl1,Mybpc, Gcnt2,MyoⅠc down-regulated;Myh4 up-regulated). The study of this part suggested that decreasing capability of learning and memory in rats might be associated with the alteration of polygene, such as up/down-regulated genes related to ion channel and neurotransmitter,signal transduction,transcription and cell structure/cytoskeleton.PARTⅣEFFECTS OF BENZ[A]PYRENE ON WHOLE HIPPOCAMPUS PROTEIN EXPRESSIONRecent years,proteomics is widely applied to study mechanisms of learning and memory.Then our study focus on investigating any possible mechanisms of B[a]P impairing learning and memory by proteomics technique(Spring’s Master Antibody Microarray).According to change of protein expression in antibody microarray,results showed that 26 proteins were up-regulated by≥1.5 folds and 23 protein were down-regulated by≥1.5 folds,when treat group compared to control.Among these,proteins perhaps associated with mechanisms of learning and memory involved: retinoic Acid Receptor b(RAR b) down-regulated≥2.6 folds,brain derived neurotrophic factor(BDNF) down-regulated≥2.1 folds, synaptotagmin iosfomrs 1(Sty1) down-regulated≥1.6 folds.The study of this part suggested that it is possible that B[a]P impair the capability of learning and memory in rats by down-regulated RAR b, BDNF and Sty1 expression.CONCLUSIONSThis study suggested that low dose of B[a]P exposure for 90 d toxicological test could impair the capability of learning and memory in rats.It is possible that impairment is related to decreasing of hippocampus amino acid neurotransmitters,NO/NOs and concentration of Ach, increasing of monoamine neurotransmitters and impairment of lipid oxidation.Decreasing capability of learning and memory in rats exposed to B[a]P might be associated with the regulation of polygene together and down-regulated RAR b,BDNF and Sty1 protein expression.It is worthy further research on concrete molecular mechanism of B[a]P impairing the capability of learning and memory in rats.更多还原