TMT对原代海马神经元损伤与BDNF保护作用研究

发布时间：2018-07-22 20:43

【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。
[Abstract]:Research background and purpose:
Three chloromethyl tin (Trimethyltin, TMT) is an important occupational hazard and an important environmental pollutant. Acute TMT exposure can cause the damage of multiple systems mainly including the nervous system damage to the central nervous system. The main mechanism of injury is to cause the apoptosis of the nerve cells, the inflammatory reaction of the nervous system, and the nervous system. .TMT can selectively induce the brain marginal system, especially the hippocampal neuron death, and cause the damage effect of the nervous system. The main role of BDNF in the central nervous system is to regulate the down regulation of BDNF expression in the rat brain of the hippocampal synaptic plasticity.TMT exposure, and the neurons that overexpress BDNF It has an antagonistic effect on the neurotoxicity of TMT. However, the damage mechanism of TMT on brain neurons, especially the damage of neuron dendrites, is not clear, and the mechanism of BDNF antagonism to the neuroprotective effect of TMT damage remains to be further studied. Therefore, this topic selects TMT and BDNF as the research content to try to understand the damage of TMT to neurons. The mechanisms and mechanisms of BDNF protection are also used to provide ideas for the treatment and prevention of neuronal damage caused by occupational exposure to TMT.
Synaptic plasticity refers to the functional enhancement or reduction of synaptic efficiency, and the changes in the intensity of neural signal transduction are accompanied by structural changes in synapses. And BDNF participates in the development and plasticity of dendritic spines that regulate activity dependent dendrites. Recent studies have shown that BDNF can be used as a regulatory factor in the adult brain to regulate neuronodes. The structure and function change.BDNF is secreted from the dendrite and immediately combines with the neurotrophic factor receptor TrkB, thereby promoting the release of the presynaptic neurotransmitter and increasing the transcriptional translation level of the postsynaptic protein, which is mainly expressed in the neuron dendrite axis, independent or in coordination with other microtubule related proteins to regulate the function of the dendritic axis microtubule. In neural cells, tau protein is associated with cell membrane or interaction with microtubule, the expression of.Tau protein or changes in structure may affect the function of its stable microtubule. Under physiological conditions, tau protein may be phosphorylated at different sites, and then affect the function of its microtubule. Mature neurons can use microtubule. The dynamic nature of the tube maintains the flexibility of the cell system to accommodate the changes occurring in the neural network.
Method:
The observation of the toxicity of TMT in the first part
18 days of primary cultured hippocampal neurons were used as a model. After treatment with TMT, the protective effect of BDNF on TMT was observed by CCK-8. The effects of TMT on the morphology of dendritic spines and the protective effect of BDNF were observed, and the characteristics of the toxicity of TMT to the dendritic spines were also identified.
The second part is the observation of the effect of BDNF on the growth of neuron synapses and dendritic spines.
The effects of the primary cultured hippocampal neurons 18 days on the synapse growth and dendritic spines in the primary cultured hippocampal neurons were observed by BDNF. The plasmids were used to transfect GFP or RFP to mark the dendritic spines of the primary hippocampal neurons. The co localization of tau egg white and microtubules was observed by cell immunofluorescence chemistry. The synapses were measured by synapses. The changes of synapse growth after BDNF treatment were measured, and Westernblot was used to detect the changes of tau protein and its phosphorylation. The changes in morphological synaptic plasticity of neurons and the role of tau protein were determined after BDNF treatment of hippocampal neurons.
The third part is the observation of Tau protein phosphorylation and cell distribution by BDNF.
RA differentiated human neuroblastoma SH-SY5Y and 18 days of primary cultured hippocampal neurons were used as models. After BDNF treatment, the subcellular distribution of tau protein was observed by cell immunofluorescence chemistry. Leica software was used to measure the synaptic growth of SH-SY5Y cells. Westernblot was used to detect the changes in the expression of tau protein, and tau eggs were determined by Westernblot. The relationship between the distribution of white subcellular cells and the synaptic growth of BDNF treated cells.
Result:
Part 1 abnormal morphological changes of dendritic spines caused by TMT
TMT treated the primary cultured hippocampal neurons 24h for 14 days in vitro, and the viability of the cells was significantly decreased by CCK-8. After the addition of BDNF and TMT, the viability of the cells was not significantly increased by CCK-8. The primary cultured hippocampal neurons were transfected to the GFP plasmid at 7 days in vitro and cultured to 20 days, TMT and / or BDNF treated the fixed cells after 24h, and the confocal microscope found TMT. After the treatment, the abnormal circle of the dendritic spines increased after BDNF treatment. The abnormal circular increase of the dendritic spines was reduced and the co stimulation of.BDNF and LiCl could also make the dendritic spines similar to the TMT treatment. The.Westerbnlot experimental results showed that BDNF treatment could be used at the same time in the primary culture. The hippocampal neurons phosphorylate Akt and ERK., but lithium alone does not seem to affect the phosphorylation of Akt and ERK. When lithium and BDNF simultaneously act, the phosphorylation of ERK induced by BDNF does not affect the phosphorylation of ERK, but lithium inhibits the abnormal increase of the dendrite dendrites induced by BDNF induced Akt phosphorylation.TMT and BDNF and LiCl. The cross-talk balance between T is damaged.
The second part of BDNF regulates synapses and dendritic spines through tau protein.
BDNF treated hippocampal neurons 24h and Tau protein expression in 14 days in vitro, and the expression of Tau protein increased significantly. The phosphorylation status of Ser262 loci was decreased after BDNF stimulation. Cell immunofluorescence chemical experiments also demonstrated that the co localization of tau protein and microtubule protein after BDNF treatment enhanced the expression of tau protein and the tree after.BDNF treatment. The density of spinous spines increased consistently. The density of dendritic spines in hippocampal neurons of the hippocampal neurons decreased significantly after 7 days in vitro culture, and the density of dendritic spines was not increased by 24h BDNF in the hippocampal neurons which were downregulated by shRNA for the 21 day after 7 days in vitro culture.
The third part of BDNF regulates the phosphorylation and subcellular distribution of tau protein.
By immunocytochemical methods, we found that tau protein forms a sphere in undifferentiated SH-SY5Y cells with a lack of neurite protuberance. On the contrary, tau protein is distributed in the neurite protuberance and the cell in the SH-SY5Y cells of 5 days of differentiation induced by retinoic acid. Through Western blot detection, we found that the treatment of retinoic acid is also Increase the level of total tau protein and decrease the expression of tau protein Ser262 phosphorylation level.Tau protein expression and the downregulation of tau protein ser262 phosphorylation level and neural cell protuberance length (related factors are r=0.94 and r=-0.98). When the original E18 hippocampal neurons were treated with microtubule depolymerization agent nocodazole, new neuron protuberance was lost. Loss of tau protein transfer to the cell body. This process of distant protrusion can be reversed to some extent by BDNF.
research conclusion
Based on the above findings, we draw the following conclusions: the abnormal increase of dendritic spines in neurons after TMT treatment in cultured hippocampal neurons may be the neurotoxic expression of TMT, the potential.TMT treatment for the protection of TMT dendritic spines and the neuron dendrite spines produced by the combination of BDNF and LiCl. Abnormal enlargement may be associated with the disruption of the cross-talk balance between ERK and Akt..BDNF can regulate the expression of tau protein in the primary hippocampal neurons. This regulation has a dose dependence and aging dependence. Transfection of the primary cultured hippocampal neurons by plasmid and down regulation of the expression of tau protein by plasmid and BDNF stimulation of the growth of dendritic spines in the neuron. Inhibition, suggesting that tau protein may be involved in BDNF signaling pathways in the growth of synapses and dendritic spines. Further experiments suggest that the role of BDNF in regulating the plasticity of dendritic spines may be achieved by regulating the expression of tau protein and thus affecting the stability of the tau protein, the phosphorylation level of the tau protein Ser262 site, the total TA. The expression of u protein and the changes in the subcellular distribution of tau protein are significantly related to the growth of synapses in the nerve cells. The increase of the expression of tau protein and the dephosphorylation of the Ser262 site may be beneficial to the growth of neural synapses. This experiment provides a new evidence for the neurotoxicity of TMT, especially on the damage of the hippocampal deity dendritic spines. It is revealed that BDNF can play a neuroprotective role by regulating the synapses and dendritic spines through tau protein.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

【共引文献】