宫内发育受限大鼠海马组织中GHSR的表达变化及其与学习记忆、情感行为的关系

河北医科大学学位论文使用授权及知识产权归属承诺本学位论文在导师(或指导小组)的指导下，由本人独立完成。本学位论文研究所获得的研究成果，其知识产权归河北医科大学所有。河北医科大学有权对本学位论文进行交流、公开和使用。凡发表与学位论文主要内容相关的论文，第一一署名单位为河北医科大学，试验材料、原始数据、申报的专利等知识产权均归河北医科大学所有。否则，承担相应的法律责任。研究生签名：赇诱 导师签章：河北医科大学研究生学位论文独创性声明本论文是在导师指导下进行的研究工作及取得的研究成果，除了文中特别加以标注等内容外，文中不包含其他人已发表或撰写的研究成果，指导教师对此进行了审定。本论文由本人独立撰写，文责自负。研究生签名：嗲碑农目 录中文摘要1英文摘要4研究论文宫内发育受限大鼠海马组织中GHSR的表达变化及其与学习记忆、情感行为的关系前言8一刖舌。材料与方法9结果20附图22附表一25讨论27结论29参考文献30综述Ghrelin及其受体GHSR与学习记忆及情感行为的关系32致谢40个人简历4l中文摘要宫内发育受限大鼠海马组织中GHSR的表达变化及其与学习记忆、情感行为的关系摘 要胎儿宫内发育受限(in仃a11terine growth retardation，IUGR)是指胎儿在宫内生长受到抑制未达到其生长潜能。临床上将此类胎儿称为小于胎龄儿(small for gestational age，SGA)，诊断标准为胎儿体重小于同孕龄平均胎儿体重的第10百分位数或小于同孕龄平均胎儿体重的两个标准差者。母体因素、胎儿及胎盘等因素如孕期营养不良、缺氧、内分泌障碍或感染等均可能导致胎儿宫内发育受限。在动物试验中，常将模型组新生幼崽平均体重低于对照组平均体重2个标准差作为造模成功的标准。IUGR引起的胎儿生长发育障碍可导致其生理、代谢等多方面的变化，与成人期多种疾病密切相关，同时可能与幼儿、学龄期儿童智力水平、性格障碍相关。由于围产期、新生儿期及儿童早期的营养、早期教育及社会环境的不同，关于IUGR对儿童神经行为发育的影响研究常常出现不同的结果。追赶生长(Catchup growth)是指当营养不良、疾病或激素缺乏时，幼儿的生长就会逐渐偏离其生长轨道，生长落后，一旦这些阻碍生长的因素被去除，幼儿将以超过相应年龄正常的速度加速生长，以恢复到原有的生长轨道上。在动物试验中也观察到宫内发育受限的幼崽的追赶生长现象。人类脑发育的关键期为孕期后3个月与生后前2年。有研究显示，宫内发育受限对脑发育存在负面影响，儿童早期生长受损可引起认知发育延迟、学习能力差，生后追赶生长中脑的快速发育可能弥补这些缺陷。生长激素促分泌素受体(Growth Hormone Secretagogues Receptor,GHSR)是在1996年从人类垂体及其他部位发现并克隆出来的一种受体，在此基础上，1999年科学家在试验鼠和人类的下丘脑弓状核及胃的内分泌细胞当中发现了一种多肽，这种物质含有28个氨基酸残基，是GHSR天然的内源性配体，具有刺激垂体释放生长激素的作用，故将该多肽物质命名为Ghrelin。Ghrelin分布于人和多种动物的中枢神经系统，如下丘脑、海马结构、丘脑区、黑质致密部、垂体、杏仁核等许多部位；在外周也有GHSR及Ghrelin的表达。大量研究显示Ghrelin及GHSR广泛参与能量中文摘要代谢、摄食行为等生理功能的调节。海马(hippocampus)是齿状回外侧，侧脑室下角底壁上的一弓状隆起，与齿状回构成海马结构。海马结构参与海马回路的构成，该环路与情感，学习和记忆等高级神经活动有关。研究发现在海马CAl区、CA2区、CA3区和齿状回等部位均发现GHSR表达。海马本身并不分泌Ghrelin，但体内其他内分泌腺产生的Ghrelin可透过血脑屏障进入海马。对于海马区Ghrelin、GHSR的深入研究发现，外源性Ghrelin促进神经细胞的修复、抑制神经细胞凋亡，同时具有调节学习记忆、情感行为的作用。但关于内源性Ghrelin水平是否直接影响上述生理过程，未见详细报道。对于海马区GHSR作用及表达调节的研究，目前尚未广泛开展。近年来有研究显示，在追赶生长后期，宫内发育受限大鼠体内的Ghrelin水平与正常大鼠无明显差异。因此我们推测，在内源性Ghrelin水平无差异，且没有外源性Ghrelin参与的情况下，GHSR表达水平的变化，可能是影响大鼠学习记忆及情感行为的因素。本研究通过建立标准的宫内发育受限大鼠模型，通过Morris水迷宫试验和抬高迷宫试验，观察宫内发育受限大鼠、追赶生长大鼠、正常大鼠之间学习记忆及情感行为的差异，并进一步采用westernblot技术及RT-PCR技术测定GHSR蛋白水平及mRNA的表达变化，探究GHSR表达的影响因素及其与学习记忆、情感行为之间的关系。目的：观察宫内发育受限大鼠及追赶生长大鼠学习记忆和情感行为变化及其海马组织GHSR的表达水平，以探讨GHSR表达的影响因素及其与学习记忆、情感行为的关系和可能机制。方法：选取2月龄成年雌性SD大鼠48只与雄性SD大鼠24只，随机选择雌雄大鼠按2：l比例合笼，使其自然交配，受孕成功的雌鼠随机分为限食组及不限食组，限食组孕鼠妊娠期均给予不限食组孕鼠饲料量的30，将该组孕鼠妊娠21天分娩，且符合条件的幼鼠随机分为两组，一组孕鼠产后继续限食(RR组)，另一组孕鼠产后不限食(RC)。孕期不限食孕鼠所产幼鼠为对照组(cc组)。分别于出生后0天，14天，28天用westernblot法测定三组大鼠海马内Ghrelin含量。并于24日龄28日龄连续5天的Morris水迷宫试验，同时于28日龄进行抬高迷宫试验，观察大鼠的学习记忆及情感行为，随后用westernblot法及RT-PCR法检测海马中文摘要内GHSR的表达。结果：1学习记忆能力RR组逃避潜伏期与RC组、CC组相比显著延长俨005)。2情感行为变化三组大鼠之间在抬高迷宫中逃避潜伏期无显著性差异(P005)，进入环绕臂的次数也无显著性差异(尸O05)。3Ghrelin的表达随着日龄的增加，在0天、14天与28天，三组大鼠海马内Ghrelin的表达量均逐渐减少；RR、RC及CC组在上述每个时间点海马内Ghrelin含量均不存在明显差异眇O05)。4GHSR的表达Westemblot检测结果：RR组GHSR表达与RC组、CC组相比均显著降低(PO01)；RC组GHSR表达与CC组相比无显著性差异pO05)；RT-PCR结果：RR组GHSR-mRNA表达量与CC组、RC组相比均显著降低愀O01)； RC组GHSR-mRNA表达量与CC组相比无显著性差异(胗O05)。结论：1宫内发育受限可能降低大鼠海马内GHSR表达水平，追赶生长可能弥补这种影响。2宫内发育受限可能影响幼鼠的学习记忆能力，追赶生长可能改善这种影响。宫内发育受限可能不是影响大鼠情感行为的因素。3宫内发育受限可能通过GHSR影响了大鼠的学习记忆能力。关键词：宫内发育受限；追赶生长；Ghrelin；生长激素促分泌素受体；学习记忆；情感行为薹奎塑墨TheexpressionofGHSRinhippocampusofintrauterinegrowthretardationratsanditsrelationshipwithmemory,learning，emorionan(1beIIavlorABSTRACTIntrauterinegrowthretardation(IUGR)isthattheintra-uterinegrowthoffetusisinhibitedwhichleadstofailureofreachingfullgrowthpotentialClinicallysuchfetusallcalledsmallforgestationalage(SGA)ThediagnosticcodeforSGAisthatthefetuswhosebirthweightislowerthanthetenthpercentileoftheaveragebirthweightcalculatedoutofthenormalfetusMaternalandfetalfactorssuchasmalnutritionduringpregnancy,ischemiaoffetusanddyscrinism，aresupposedtobepossibleelementscausingIUGRDuringtheprocessofanimalexperiment，thetargetofmodelingIUGRanimalsisthatbirthweightofnewbornsinmodelgroupislowerthanx一2sdofthatincontrolgroupIUGRCanleadtochangesinphysiology,metabolism，whicharecloselyrelatedtomanydiseasesinadultstageIUGRisalsosupposedtoplayaroleintheintellectualdevelopmentandtheformationofcharacterdisorderinchildrenDuetothechangesinperinatal-neonatalfactors，educationfactors，nutritionconditionsandsocialenvironment，researchesconcerningtheeffectsofIUGRonneurobehavioraldevelopmentofchildrenoftenresultsindifferentoutcomesWhensufferedmalnutrition，illness，lackofnecessaryhormone，childrengraduallydeviatefromtheiroriginalgrowthrhythm，whichleadstodevelopmentalretardationOncethefactorsmentionedaboveareremoved，childrenSgrowthwouldacceleratetorestorethelossThisphenomenonisdefinedasCatchupgrowth(CUG)CatchupgrowthhasalreadybeenobservedinIUGRanimalmodelsResearchshowsthatIUGRhasanegativeinfluenceonbraindevelopmentwhichleadstodelayofcognitionandlearningdysfunction，whilecatch-upgrowthcouldcompensatefortheproblemsmentionedabove4英文摘要Growth Hormone Secretagogues Receptor(GHSR)is a protein that isdiscovered in hypothalamus and other kind of tissue inside human body andsuccessfully cloned in 1 996On basis of that，scientists discovered apolypeptide named Ghrelin that contains 28 aminoacid residuesT11ispolypeptide was found in nucleus arcuatus hypothalami and stomach of bothhuman and ratItS an endogenous ligand for GHSRGhrelin distributes inmany part of central nervous system and peripheral tissues，both in human andnonhuman animalsIt has a stimulus effect on hypophysis which leads tosecretion of growth hormoneA lot of work showed Ghrelin and GHSR has awideranging impact on regulating physiological functions such as energymetabolism and feeding behaviorHippocampus is a structure in central neural systemItS an arcuateeminence outside the dentate gyrus and is located on bottom of inferior comuof lateral ventricleHippocampus and the dentate gyrus make up thehippocampus structureIt participates in many advanced neural actions such asthe formation of behavior,emotion，learning and memoryResearches showmat GHSR distributes in CA l district，CA2 district，CA3 district andespecially the dentate gyrusHippocampus itself does not secrete Ghrelin，but一 一_ 一一 Ghrelin secreted by other ligands is able to go through the bloodbrain barrier(BBB)into the hippocampusFurther research on Ghrelin and GHSR showedthat exogenous Ghrelin promotes the reparation of hippocampus nerve cells，inhibits apoptosis of hippocampus neural cellsIt also regulates learning，memory,emothion and behaviorHowever,no reports show endogenousGhrelin could influence those physiological processes mentioned aboveResearch on effect of GHSR and regulating mechanics of GHSR expression inhippocampus has not yet been widely carried outIn recent years，research shows that in the later stage of catchup growth，Ghrelin level in IUGR rats has no significant difference compared with normalonesThus we could predict that、析m the same endogenous Ghrelin level andthe absence of exogenous Ghrelin，the expression level of GHSR could be thepossible factor on regulating memory,learning，emotion and behavior in rats5英文摘要This experiment would build an IUGR rat model with food restriction duringpregnancyRats are divided into IUGR group，catchup growth group andcontrol groupWe would evaluate the changes of learning，memory,emotionand behavior between these three groups，using elevated plus maze test andMorris water-maze testFurthermore，we would detect the changes of GHSRexpression with westernblot，check the expression of GHSR-mRNA withRT-PCR，through which two methods to explore the relationship between thefactors that influence GHSR expression and the physiological functionsmentioned aboveObjective：To observe the changes of learning，memory,emotion andbehavior in IUGRcatchup growth rats，detect GHSR expression inhippocampus of such ratsTo discuss the relationship between the factors thatinfluence GHSR expression and the physiological functions mentioned aboveand explore the possible mechanicsMethods：48 healthy female adult SD rats and 24 health male adult SDrats are randomly put in to 24 cages in portion of 2：1Then rats mate naturallyPregnant rats are randomly divided into 3 groups：food restriction throughoutpregnancy and lactation period；food restriction during pregnancy and no foodrestriction during lactation period；no food restriction throughout pregnancyand lactation periodJuvenile rats who meet the standards are respectively putinto RR group(IUGR group)，RC group(Catch-up growth group)and CCgroup(control group)The content of Ghrelin in hippocampus of each groupis detected at day 0，day 1 4，day 28Elevated plus maze test are carried out atday 28 and Morris water-maze test are carried out from day 24 to day 28 inorder to observe the changes of learning，memory,emotion and behaviorGHSR expression in hippocampus is detected wim western-blot andGHSR-mRNA iS detected wim Rr-PCRResults：1Learning and memory Compared with both RC group and CCgroup，escape latency of RR group significantly lengthened(P00 1)，thenumber of times of crossing the platform significantly decreased俨005)2Emotion and behavior There are nosignificant difference in escape latency and numbers of time of visiting theclosed arms between the 3 groups in elevated plus maze test(P005)3Expression of Ghrelin Western-blot shows that expression of Ghrelin inhippocampus in each group decreased graduallyThere are no significant