【作者】 李晓恒；

【导师】 刘能保；

【作者基本信息】 华中科技大学 ， 人体解剖学和组织胚胎学， 2007， 博士

【摘要】 慢性应激对学习与记忆功能的影响已成为当前神经科学研究领域的热点课题之一。海马结构是参与学习记忆功能的关键脑区,又是应激反应的靶部位。因此应激所致的海马相关功能障碍必定影响学习记忆功能,但已有研究发现,不同的应激刺激对学习和记忆的影响不同,并且存在着争议。诸多研究报道慢性躯体性、心理性或复合应激都可能导致海马神经元发生形态学改变,甚至死亡,从而影响脑的认知功能,坏动物的学习与记忆能力。也有少数实验结果显示慢性复合应激对学习与记忆功能产生增强作用。以往的慢性应激导致记忆受损的实验动物模型通常采用单一应激原,例如捆绑、电击、致痛,睡眠剥夺或噪音等。本课题的创新之处,在于采用由4种应激原组成的复合应激,建立有效的慢性复合应激(chronic multiple stress)动物模型,进行以下实验。①观察慢性复合应激6 w后大鼠学习和记忆的变化情况。②观察慢性复合应激6 w后海马内突触素(ISynapsin I),非受体酪氨酸激酶Fyn,脑源性神经营养因子(BDNF)和受体型酪氨酸激酶Trk酪氨酸激酶B (TrkB)的蛋白表达变化。③观察慢性复合应激6 w后海马内Fyn mRNA, TrkB mRNA水平的变化。④通过对慢性复合应激大鼠进行侧脑室注射酪氨酸激酶抑制剂PP2,观察其对慢性复合应激性学习记忆增强大鼠学习和记忆的影响,以及海马内Fyn, BDNF, TrkB蛋白表达的改变。⑤从离体水平,即通过海马神经元的原代培养,向培养基中加入酪氨酸激酶抑制剂PP2,观察其对海马神经元胞浆内钙离子浓度([Ca2+]i)和Fyn表达水平的影响。本文从在体水平和离体水平所获得的结果是一致的。本实验分为三部分:第一部分:探讨慢性复合应激对大鼠学习与记忆的作用和海马内突触素I(Synapsin I),非受体酪氨酸激酶Fyn,脑源性神经营养因子(BDNF)和Trk酪氨酸激酶B(TrkB)的蛋白及Fyn mRNA, TrkB mRNA水平的变化及其在学习与记忆中的作用。将成年雄性Wistar大鼠分为对照组及慢性复合应激组,后者采用垂直旋转、睡眠剥夺、捆绑和光照4种应激原无规律交替刺激达6 w。实验过程中每周一次测定动物体重的变化。应激试验前用Morris水迷宫(MWM)连续3d测试两组动物的学习记忆成绩,应激试验结束后,两组动物再进行MWM测试,并将动物分为以下3组:①形态学观察组,使用免疫组织化学技术观察Synapsin I, Fyn, BDNF在海马CA1区、CA3区和齿状回(dentate gyrus,DG)区的分布和表达;运用尼氏染色方法,观察海马结构各亚区神经细胞密度的变化;②Western Blotting测定组,通过对海马组织总蛋白的提取,测定海马总蛋白中Fyn, BDNF, TrkB的表达水平的变化;③RT-PCR测定组,通过对海马组织总RNA的提取,测定Fyn和TrkB mRNAs水平的变化。结果显示:动物经过6 w应激后,①MWM测试结果显示,慢性复合应激组动物的学习记忆成绩优于对照组;②免疫组织化学观察分析表明,Synapsin I免疫反应阳性产物,分布于树突和轴突内;Fyn的免疫产物主要分布在海马的CA3区辐射层(树突);在海马CA1区、CA3区和DG区内可见BDNF免疫反应阳性产物主要分布在锥体细胞和颗粒细胞的细胞核内,并且,应激组动物海马组织中Synapsin I, Fyn, BDNF蛋白表达量均增加;③慢性复合应激组海马CA1、CA3及DG区的神经细胞密度明显增高;④Western Blotting结果显示,慢性复合应激组海马总蛋白中Fyn, BDNF和TrkB的表达均增加;⑤RT-PCR的结果显示:慢性复合应激组海马Fyn mRNA水平上调。上述结果提示:慢性复合性应激导致大鼠学习与记忆能力增强,海马结构各亚区神经元密度以及在海马组织中Synapsin I, Fyn, BDNF, TrkB蛋白以及Fyn的mRNA水平增加,提示Fyn和BDNF/TrkB信号途径参与了慢性复合应激性学习记忆增强的机制。第二部分:进行侧脑室注射酪氨酸激酶抑制剂PP2,观察酪氨酸激酶抑制剂PP2对慢性复合应激性学习记忆增强大鼠的学习记忆功能和海马内非受体酪氨酸激酶Fyn,脑源性神经营养因子(BDNF)和Trk酪氨酸激酶B(TrkB)蛋白表达的影响。成年雄性大鼠随机分为慢性复合应激对照组(对照组)、慢性复合应激+注射盐水组(盐水组)和慢性复合应激+注射PP2组(PP2组),全部动物暴露于复合应激原中6 w后,盐水组和PP2组动物分别侧脑室注射生理盐水或PP2抑制剂(1次/天,共11天)。实验结束后,用MWM测试大鼠的空间学习记忆成绩;采用免疫组织化学方法检测Fyn, BDNF和TrkB在海马内蛋白表达的变化。结果显示:①与对照组和盐水组相比,PP2组动物的学习与记忆成绩明显下降;②海马内Fyn和BDNF蛋白阳性表达减弱;③3组动物海马内TrkB的蛋白表达无显著差异。以上结果表明,侧脑室注射PP2抑制大鼠慢性复合应激性学习记忆能力的增强作用,下调Fyn和BDNF在海马内的表达。Fyn以及BDNF/TrkB信号转导途径在慢性复合应激增强大鼠学习记忆能力的过程中发挥重要作用。第三部分:探讨酪氨酸激酶抑制剂PP2对原代培养的海马神经元及其胞浆内钙离子浓度([Ca2+]i)和海马神经元非受体酪氨酸激酶Fyn蛋白表达的影响和可能的机制。取生后24～48 h Wistar乳鼠的海马组织进行神经元原代培养,培养10 d后,使用抗神经元特异性烯醇化酶(neuron specific enolase,NES)抗体测定海马神经元纯度,并且将细胞分为对照组(只加无血清培养基)和终浓度为12.5μmol/L PP2的PP2处理组,继续培养4 d,进行如下实验:①观察不同培养时间,海马神经元形态学的变化;②观察PP2对海马神经元形态学的影响;③应用AnnexinV-PI试剂盒,结合流式细胞术,观察PP2导致海马神经元凋亡情况;④采用激光共聚焦显微镜技术,对两组组海马神经元[Ca2+]i进行测定;⑤通过细胞免疫化学和Western Blotting技术检测PP2导致海马神经元Fyn分布和表达的变化。结果显示:①海马神经元贴壁及时,生长良好,且能够形成神经网络;②本实验培养的海马神经元的纯度达到70%;③PP2对海马神经元的形态具有影响,PP2处理组海马神经元突起回缩,神经网络稀疏;并且可引起海马神经元的早期凋亡;④PP2可引起海马神经元[Ca2+]i水平的增高;⑤免疫细胞化学以及Western Blotting的结果显示,加入PP2干预因素后,海马神经元Fyn表达呈现下降的趋势,海马神经元[Ca2+]i水平和海马神经元Fyn蛋白表达呈现负相关。上述结果提示,在PP2的作用下,海马神经元出现早期凋亡。海马神经元[Ca2+]i水平升高可能既是PP2导致海马神经元损伤的结果,又是引起海马神经元发生凋亡和Fyn表达下降的原因。总而言之,本实验从整体动物水平和离体细胞水平两方面着手,观察了慢性复合应激动物和原代培养海马神经元各相关指标的变化,两者既是相互独立,又通过酪氨酸激酶抑制剂—PP2紧密地联系在一起。慢性复合应激大鼠学习和记忆能力增强,海马各区细胞数量增多,海马内Fyn, BDNF和TrkB的表达也增高;侧脑室注射PP2抑制大鼠慢性复合应激性学习记忆能力的增强作用,下调Fyn和BDNF在海马内的表达;在离体培养的海马神经元中,培养基中加入的酪氨酸激酶抑制剂PP2,海马神经元形态受到影响,出现早期凋亡,海马神经元[Ca2+]i水平升高以及Fyn表达降低。Fyn以及BDNF/TrkB信号转导途径在慢性复合应激增强大鼠学习记忆能力的过程中发挥重要作用。更多还原

【Abstract】 The effect of chronic stress on learning and memory functions has been one of the hot topics in neuroscience. Hippocampus is a key region highly involved with leaning and memory and it is the target site of stress. Therefore,stress induced functional impairment of the hippocampus must impact the learning and memory function. But the investigations found that different stress stimulation has different effect to learning and memory,and there has been much controversy over its mechanism. Many studies show that chronic body,mental,or multiple stress can leads to the morphology changes,even the death of the hippocampal neuron. Meanwhile it impacted the brain cognitive functions and induced to learning and memory impairment. There are a few studies indicated that chronic multiple stress could enhance the learning and memory function. Such single stressor as restraint,foot shock,induced pain,sleeping deprivation or noise,which impaired learning and memory has been applied to make animal models in the past. It is an innovation of this topic that animals were given chronic multiple stresses formed by four kinds of stressors at the same time to perform the following experiments:①Observe the changes of rats’learning and memory after six-week stresses.②Observe the expression changes of Synapsin I,Fyn,BDNF and TrkB protein levels after six-week stresses.③Observe the changes of Fyn mRNA and TrkB mRNA levels after six-week stresses.④The rats were injected PP2,a tyrosine kinase inhibitor in lateral cerebral ventricle to study the effects of PP2 on function of learning and memory and the expression of Fyn,BDNF,TrkB in the hippocampus of the rat whose leaning and memory was enhanced by chronic multiple stress.⑤In addition,PP2 was added to culture media to observe the effect of PP2 on the free calcium concentrations ([Ca2+]i) and Fyn expressions in hippocampal neurons by culturing primarily hippocampal neurons in vitro. The results in vivo were in agreement with those in vitro.This experiment includes three parts:Part I. The objective of the first part is to investigate the effects of chronic multiple-stress on learning and memory and on the levels of Synapsin I、Fyn, BDNF, TrkB protein and Fyn, TrkB mRNAs.Adult male Wistar rats were randomly divided into control and chronic multiple-stressed groups. The latter were administered four kinds of stressors,including vertical rotation,sleeping deprivation,restraint and illumination irregularly and alternately for 6 weeks. The body weights were examined once every week during the period of the stress. In order to examine the scores of leaning and memory,two groups of animals were given 3-day Morris Water Maze (MWM) test before the stress and MWM tests successively after completing the stress. The animals were divided into the following three groups:①Morphological group:Observe the distributions and expressions of Synapsin I,Fyn and BDNF in the hippocampal CA1,CA3 and DG using immunohistochemical technique. Examine the nerve cell densities of hippocampal CA1,CA3 and DG using Cresyl violet stain.②Western Blotting groups: Measure the Fyn,BDNF,TrkB protein expressions of hippocampal total protein extracted.③RT-PCR groups:Measure the Fyn mRNA and TrkB mRNA levels of the total mRNA isolated from hippocampus.These results were showed as follows: After stress,①Learning and memory scores of chronic multiple-stressed rats excelled those of the control group in the MWM.②The results of mmunohistochemical technique indicated that Synapsin I , Fyn and BDNF-immunostaining of the chronic multiple-stressed rats was harder than that of the control group. Respectively,the distributions of them are in the dendrites and axons of the pyramidal and granule cells of hippocampal (synapsin I),or in the stratum radiatum of hippocampal CA3(Fyn),or in the nucleus of the pyramidal and granule cells of hippocampal CA1,CA3,DG (BDNF).③The nerve cell densities in hippocampal CA1,CA3 and DG of chronic multiple-stressed animals were markedly higher than that of the control group.④The Fyn,BDNF,TrkB proteins of hippocampal total protein in chronic multiple-stressed group enhance significantly as compared with the control one from the Western Blotting.⑤The Fyn mRNA levels in chronic multiple-stressed group rose significantly as compared with the control one too from the RT-PCR.These results maked clear that chronic multiple stress can enhance spatial learning and memory function of rats. The cell density of hippocampal subregions is increased in the stessed rat. The expression of Synapsin I,Fyn,BDNF and TrkB proteins and the level of Fyn mRNA are increased in the stessed rat hippocampus respectively. Its suggest that BDNF/ TrkB signal transduction pathway and Fyn may participate in the process of the enhanced learning and memory by chronic multiple stress.PartⅡ. The second part aimed to investigate the effects of PP2,a tyrosine kinase inhibitor,on function of learning and memory and the expression of Fyn,BDNF,TrkB in the hippocampus of the rat whose leaning and memory was enhanced by chronic multiple stress with the method of lateral cerebral ventricle injecting.Adult male rats were randomly divided into three groups:chronic multiple stressed group (control group),chronic multiple stressed with saline group (saline group) and chronic multiple stressed with PP2 group (PP2 group). All rats were exposed to chronic multiple stress for 6 weeks,then rats of saline group and PP2 group were injected normal saline or PP2 in lateral cerebral ventricle(11d with one time / d). After that,the performance of spatial learning and memory of all rats was measured using Morris water maze. The expression of Fyn,BDNF and TrkB proteins in the hippocampus of rats were detected by immunohistochemical method.The Results showed:①Compared with control group and saline group,the performance of spatial learning and memory of rats was decreased in PP2 group;②The immunoreactivities of Fyn and BDNF in the hippocampus were weakened in rats of PP2 group;③But no difference of TrkB protein level in the hippocampus was observed between the three groups.These results maked clear that PP2 with intracerebro-ventricular may degrade the function of rat’s learning and memory,and downregulated expression of Fyn and BDNF in the hippocampus of the rat whose leaning and memory was enhanced by chronic multiple stress. It suggests that Fyn and BDNF/TrkB signal transduction pathway may participate in the process of the enhanced learning and memory by chronic multiple stress.PartⅢ. The third part aimed to explore the effects of PP2,a tyrosine kinase inhibitor,on primary cultured hippocampal neurons and their free cytoplasmic calcium concentration ([Ca2+]i ) and Fyn expressions and its’possible mechanisms.The hippocampi were taken from 24 to 48-hour postnatal Wistar rats and hippocampal neurons were primarily cultured. 10 days later,the purity of neurons was detected using the antibody to neuron specific enolase (NSE) and the cells were divided into control group (with only serum-free DMEM-F12) and groups treated with PP2,which final concentration is 12.5μmol/L. All groups were performed the following experiments after the cells were cultured for 4d;①Observe the morphological changes of hippocampal neurons in different time;②Observe the morphological changes of hippocampal neurons in control group and PP2 group;③Explore the apoptosis of hippocampal neurons induced by PP2 using annexinV/PI by flow cytometry;④Measure the hippocampal neurons [Ca2+]i of two groups under the confocal laser microscope;⑤Examine the distributions and expressions ofFyn in hippocampal neurons,treated with PP2 by adopting the method of immunocytochemistry and Western blotting.These results were showed as follows:①The hippocampal neurons could adhere to the bottom of culture plate in time,grow well and form neural network.②Purity of hippocampal neurons in this experiment exceeded 70%.③The morphological change of hippocampal neurons was appearanced in PP2 group. Compared with the control neurons,the neurite of hippocampal neurons retracted and the neural network became rare. The primary apoptosis was caused in the PP2 groups.④There was a marked increase in hippocampal neurons [Ca2+]i at the PP2 group.⑤Fyn expressions were decreased in hippocampal neurons with PP2 in the immunocytochemistry and Western blotting technique. Their differences were statistically signifcant. There was a negative correlation between hippocampal neurons [Ca2+]i and Fyn expressions.The primary apoptosis occurred in the hippocampal neurons treated with PP2. The increase of hippocampal neurons [Ca2+]i might both result from an impairment of the hippocampal neurons induced by PP2 and result in the apoptosis of hippocampal neurons and low expressions of Fyn.To sum up,the changes in these indexes of chronic multiple-stressed rats and primarily cultured hippocampal neurons were observed at the whole and cellular level using in-vivo and vitro model system in this study. They were both independent and closely interrelated depending on PP2,a tyrosine kinase inhibitor. Chronic multiple stress can enhance spatial learning and memory function of rats. The cell density of hippocampal subregions is increased in the stessed rat. The expression of Fyn,BDNF and TrkB proteins and the level of Fyn mRNA are increased in the stessed rat hippocampus respectively. PP2 with intracerebro-ventricular may degrade the function of rat’s learning and memory,and downregulated expression of Fyn and BDNF in the hippocampus of the rat whose leaning and memory was enhanced by chronic multiple stress. The morphological change of hippocampal neurons was appearanced in PP2 group on primary cultured hippocampal neurons. Primary apoptosis was caused and there was a marked increase in hippocampal neurons [Ca2+]i at the PP2 group. Fyn expressions were decreased in hippocampal neurons with PP2. Fyn and BDNF/ TrkB signal transduction pathway may participate in the process of the enhanced learning and memory by chronic multiple stress.更多还原