【作者】 陈前；

【导师】 余争平；

【作者基本信息】 第三军医大学 ， 环境与劳动卫生学， 2014， 博士

【摘要】 研究背景和目的：三氯甲基锡(Trimethyltin,TMT)是一种重要的职业危害毒物，也是一种重要的环境污染物。急性TMT暴露可引起多系统主要包括神经系统的损害。TMT作用于中枢神经系统，主要损伤作用机制为引起神经细胞凋亡，引起神经系统炎性反应，对神经组织造成氧化损伤等。TMT可以选择性地诱导大脑边缘系统，特别是海马神经元死亡，造成神经系统损伤效应。研究表明，BDNF在中枢神经系统的主要作用是调节海马神经突触可塑性。TMT暴露的大鼠大脑BDNF表达下调，而过表达BDNF的神经元具有拮抗TMT神经毒性的作用。但TMT对大脑神经元的损伤机制特别是对神经元树突棘的损伤研究还不清楚，并且BDNF拮抗TMT损伤发挥神经保护作用的机制尚待进一步研究。因此，本课题选取TMT及BDNF为研究内容，试图理解TMT对神经元的损伤作用机制及BDNF保护作用的分子机制，并为治疗和预防TMT职业暴露造成的神经元损伤提供思路。突触可塑性指突触效率的功能性增强或降低，同时神经信号传递强度的变化伴随着神经元突触的结构变化。并且，BDNF参与调节活动依赖的树突棘发育及可塑性。近期研究表明BDNF可以作为成年脑中活动依赖性调节因子参与调控神经结构和功能变化。BDNF从树突分泌出来后，立即与神经营养因子受体TrkB结合，进而促进突触前神经递质的释放并且增加突触后蛋白的转录翻译水平。Tau蛋白主要表达于神经元树突轴，独立或与其他微管相关蛋白协同调节树突轴微管功能。在神经细胞中，tau蛋白与细胞膜相联系或与微管发生相互作用。Tau蛋白表达或者结构的变化可能影响其稳定微管蛋白的功能。在生理条件下，tau蛋白可能在不同的位点发生磷酸化改变，进而影响其稳定微管的功能。成熟神经元可以利用微管的动态性保持细胞体系的弹性，以适应神经网络发生的各种变化。方法：第一部分TMT对神经元毒性的观察以原代培养的胚胎期18天海马神经元为模型，用TMT处理后，用CCK-8的方法观测BDNF对TMT的保护作用，观察TMT处理后神经元树突棘形态的影响以及BDNF的保护作用，并明确TMT对神经元树突棘毒性的特征；第二部分BDNF对神经元突触和树突棘生长影响的观察以原代培养的胚胎期18天海马神经元为模型，用BDNF处理，观察对原代培养海马神经元突触生长和树突棘形态的影响；利用质粒转染GFP或RFP标记原代海马神经元树突棘，细胞免疫荧光化学观测tau蛋白和微管的共定位情况，突触综合体测量BDNF处理后突触生长的变化，westernblot检测tau蛋白及其磷酸化变化，明确BDNF处理海马神经元后，神经元的形态突触可塑性的变化以及tau蛋白所起的作用；第三部分BDNF对Tau蛋白磷酸化及细胞分布的观察以RA分化的人神经母细胞瘤SH-SY5Y和原代培养的胚胎期18天海马神经元为模型，用BDNF处理后，采用细胞免疫荧光化学的方法观察tau蛋白的亚细胞分布情况，Leica软件测量SH-SY5Y细胞的突触生长情况，westernblot检测tau蛋白表达变化,明确tau蛋白亚细胞分布和BDNF处理后细胞的突触生长的关系；结果：第一部分TMT引起树突棘的异常形态改变TMT处理体外培养14天的原代海马神经元24h，CCK-8检测细胞活力显著下降；加入BDNF与TMT共刺激后，CCK-8检测细胞活力无显著上升。原代海马神经元体外培养7天时转染GFP质粒，培养至20天，TMT和/或BDNF处理24h后固定细胞，共聚焦显微镜发现TMT处理后神经元树突棘出现异常圆形增大。加入BDNF处理后，树突棘的异常圆形增大有减少的趋势。BDNF与LiCl共刺激能够同样使神经元树突棘出现与TMT处理后高度相似的异常圆形增大。Westerbnlot实验结果表明，BDNF处理后可以同时在原代培养的海马神经元磷酸化Akt和ERK。而锂单独似乎不能影响Akt和ERK的磷酸化状态。当锂与BDNF同时作用时，BDNF诱导的ERK的磷酸化没有影响，但锂却抑制BDNF诱导的Akt磷酸化。TMT以及BDNF和LiCl联合作用产生的神经元树突棘异常增大可能与ERK与PI3K-Akt之间的cross-talk平衡被破坏有关。第二部分BDNF通过tau蛋白调节突触和树突棘生长BDNF处理体外培养14天的海马神经元24h，Tau蛋白的表达都显著升高，Ser262位点的磷酸化状态在BDNF刺激后具有下降的趋势。细胞免疫荧光化学实验也证明tau蛋白与微管蛋白在BDNF处理后的共定位得以增强。BDNF处理后tau蛋白的表达变化与树突棘密度的增加一致。运用shRNA技术在体外培养7天时下调tau蛋白表达水平后，培养至21天海马神经元树突棘密度显著下降。并且在shRNA下调tau蛋白的海马神经元中，24h BDNF刺激不能增加神经元的树突棘密度。第三部分BDNF调节tau蛋白的磷酸化与亚细胞分布通过免疫细胞化学方法，我们发现在未分化的缺乏神经突起的SH-SY5Y细胞中，tau蛋白形成一个球体。相反，在维甲酸诱导分化5天的SH-SY5Y细胞中，tau蛋白分散分布在神经细胞突起和胞体中。通过Western blot检测方法，我们发现维甲酸的处理也增加了总tau蛋白水平而降低tau蛋白Ser262磷酸化水平。Tau蛋白表达上调和tau蛋白ser262磷酸化水平的下调与神经细胞突起长度呈相关性(相关因子分别为r=0.94和r=-0.98)。当原E18海马神经元用微管解聚剂nocodazole处理后，新生的神经元突起丢失并且发生tau蛋白转移到胞体的现象。这种远离突起的过程可以被BDNF一定程度上逆转。研究结论基于以上研究结果，我们得出以下研究结论：TMT处理体外培养的海马神经元后发现神经元的树突棘出现异常的增大现象，可能是TMT的神经毒性表现。BDNF具有保护TMT树突棘损伤的潜力。TMT处理以及BDNF和LiCl联合作用产生的神经元树突棘异常增大可能与ERK与Akt之间的cross-talk平衡被破坏有关。BDNF能够调节原代海马神经元tau蛋白的表达,这样的调节具有剂量依赖关系和时效依赖关系。通过质粒转染原代培养海马神经元，下调tau蛋白表达，BDNF刺激神经元树突棘生长的现象被抑制，说明tau蛋白可能参与了BDNF调节神经元突触及树突棘生长的信号通路。进一步实验表明，BDNF调节树突棘可塑性的作用可能是通过调节tau蛋白的表达量进而影响tau蛋白稳定微管能力来实现的；tau蛋白Ser262位点的磷酸化水平，总tau蛋白的表达量以及tau蛋白的亚细胞分布变化与神经细胞突触生长的显著相关，tau蛋白的表达增加以及Ser262位点的去磷酸化可能以利于神经细胞突触的生长。本实验为TMT的神经毒性特别是对海马神经元树突棘的损伤提供新的证据，也揭示了BDNF可以通过tau蛋白调节突触以及树突棘生长发挥神经保护作用。更多还原

【Abstract】 BackgroundTMT is an important occupation poison, and is a risk factor of environmental pollution.Acute TMT exposure could cause multi system including nerve system damage. The maindamage caused by TMT in the central nervous systemincludes apoptosis of nerve cells,neuronal inflammation and oxidative damage of the nervous tissue. TMT can selectivelyinduce the limbic system, especially caused the hippocampal neuronal death effect. BDNFplays a main role in regulating hippocampal synaptic plasticity in the central nervoussystem. Rat brain exposed to TMT showed reduced BDNF expression and the neurons overexpression of BDNFcould antagonist the neurotoxicity of TMT. But the mechanism ofTMT damage to the brain neurons especially the damage to neuronal dendritic spines isunclear, and the mechanisms of BDNF antagonist TMT damage effect remains to be furtherstudied.Therefore, this paper chooses BDNF and TMT as the research content, trying tounderstand TMT damage to the neural synaptic plasticity and the molecular mechanism ofBDNF protective effect, in order to provide clues for the treatment and prevention of TMToccupation exposure on synaptic plasticity damage.Synaptic plasticity refers to the functional synaptic efficiency enhanced or reduced, atthe same time transmission of neural signal intensity changes accompanied by structuralchanges in neuronal synapse. Moreover, brain derived neurotrophic factor involved in thedevelopment and plasticity of dendritic spines of regulating activity dependent. Recentstudies show that BDNF can be used as the adult brain activity dependent regulation offactors involved in the changes of structure and function of regulating nerve. BDNFsecreted from the dendrites, immediately combined with neurotrophic factor receptor TrkB,and then promote the presynaptic neurotransmitter release and increase the level oftranscription and translation of the postsynaptic protein. Tau protein was mainly expressedin the neuronal dendritic shaft, independent or associated with other tubulin co regulation ofdendritic shaft microtubule function. In nerve cells, tau protein and cell membrane associated or interact with microtubules. Expression of Tau protein or structure may affectthe stability of microtubule protein function. Under physiological conditions, tau proteinphosphorylation changes may occur at different sites, thereby affecting its microtubulestabilizing function. Mature neurons can use dynamic microtubules maintain cell system, toadapt to the changes of neural network place.MethodsPart I: To observe the neurotoxity of TMTPrimary cultures of embryonic day18hippocampal neurons were used as model. Afterthe treatment with TMT, the protective effect of BDNF was observed by CCK-8assay;neuronal dendritic spine morphology were observed after treatment with TMT and TMTplus BDNF.We would like to address the characteristic of TMT toxicity on dendritic spine.Part II: To observe the effect of BDNF in the regulating synaptic plasticityPrimary cultured embryonic day18(E18) hippocampal neurons of embryonic day18was treated by BDNF, the effect of the hippocampal synaptic growth and dendritic spineswas observed; GFP, RFP plasmids were transfected into primary hippocampal neurons tolabel dendritic spines; colocalization of tau protein and the microtubules was examined byimmunocyochemistry; synaptic complex was measured after treatment with BDNF; tauprotein expression and phosphorylation was detected by Westernblot. These experimentsare set to investigate the role of tau protein in neuronal morphology changes in synapticplasticity of hippocampal neurons.Part III: To observe the effect of BDNF on Tau phosphorylation and celldistributionRetinoic acid (RA) differentiated human neuroblastoma SH-SY5Y and primary cultureof embryonic day18hippocampal neurons were used as a model. After the treatment withBDNF, the subcellular distribution of tau protein was observed by immonocytochemistry;SH-SY5Y cell outgrowth was measured by Leica software; expression of tau protein wasdetected by Westernblot. These experiments were set to address the possible relationshipbetween the cellular distribution of tau protein and BDNF treatment during cell outgrowth.Results：Part I: TMT could induce abnormal morphology of dendritic spines 14DIV Primary cultured hippocampal neurons were treated by TMT for24h; cellviability was decreased significantly by CCK-8assay; while the application of BDNFtogether with TMT, cell viability does not increase significantly. Primary culturedhippocampal neurons were transfectd of GFP plasmid at7DIV and then were cultured to20days. Then it was treated by TMT and/or BDNF for24h. Confocal microscopy showedthat TMT treatment could cause the occurrence of ball-like morphological abnormalities ofdendritic spine. The application of BDNF could reduce the morphological abnormalities ofdendritic spine to some extent. BDNF and LiCl co-stimulation can also mimic this dendriticspine abnormality, which share great similarity of that treated by TMT. Westerbnlotshowed that BDNF treatment could both phosphorylated Akt and ERK in primary culturedhippocampal neurons (Figure2). Lithium alone does not seem to affect the phosphorylationstatus of both Akt and ERK. When adding the lithium together with BDNF, thephosphorylation of ERK induced by BDNF was not affected. But lithium inhibited thephoisphorylation of Akt induced by BDNF. TMT induced abnormal enlargement ofdendritic spine as well as that induced by BDNF and LiCl cotreatment might result from theimbalance of the cross-talk between ERK and Akt.Part II: BDNF regulating morphological synaptic plasticity through tau protein14day in vitro (DIV) hippocampal neurons were treated by BDNF for24h. Tauprotein expression was significantly increased and phosphorylation at Ser262wasdecreased after BDNF stimulation. Immunocytochemistry showed that colocalization of tauprotein and tubulin was enhanced by BDNF treatment. The increased tau protein expressionwas also consistent with the increase of dendritic spine density after BDNF treatment.Application of shRNA methods in cultured neurons at7days to down-regulate tau proteinexpression level, and then we observe at21day that hippocampal dendritic spine densitywas decreased significantly. And in hippocampal neurons whose tau protein was thedown-regulated by shRNA plasmid,24h BDNF stimulation could not increase the densityof dendritic spines of neurons.Part III: BDNF regulation of tau protein phosphorylation and subcellulardistributionBy immunocytochemical method, we found that in undifferentiated SH-SY5Y cellslacking neurites, tau protein could form a sphere. In contrast, in SH-SY5Y cells differentiated by retinoic acid for5days, tau protein was distributed in the neuronalprocesses and the cell body. Through the Western blot detection method, we found thatretinoic acid also increased total tau protein levels and decreased tau protein levels ofSer262phosphorylation. The up-regulated expression of tau protein and tau proteinphosphorylation level of ser262reduction was associated with the neuronal processes (R=0.94and R=-0.98). When the primary cultured E18hippocampal neurons were treated withthe microtubule depolymerizing agent nocodazole, neuronal loss occurred and new tauprotein was transferred to the cell body. This process can be reversed the application ofBDNF to some extent.Conclusion：Based on the above results, we can draw the following conclusions that: TMTtreatment could cause the occurrence of ball-like morphological abnormalities of dendriticspine, which might be an important character of TMT neurotoxicity. The application ofBDNF showed potential protection against this damage. TMT induced abnormalenlargement of dendritic spine as well as that induced by BDNF and LiCl cotreatmentmight result from the imbalance of the cross-talk between ERK and Akt. BDNF canregulate the expression of tau protein in primary hippocampal neurons, and tau protein maybe involved in the signal pathway of BDNF regulating the synaptic plasticity, and theregulation effect may be achieved through regulation of tau protein expression andmodulation of tau protein microtubule stabilizing ability; tau protein Ser262sitephosphorylation, total tau protein expression and subcellular distribution of tau proteinchanges were closely related to the neuronal outgrowth; Our experiments provide newevidence for the neurotoxicity of TMT to hippocampal neurons, and also provide a newclue in revealing the role of tau in BDNF signaling pathway in regulating neuronaloutgrowth and dendritic spine morphology.更多还原