TMT對原代海馬神經(jīng)元損傷與BDNF保護(hù)作用研究
發(fā)布時間：2018-07-22 20:43
【摘要】：研究背景和目的： 三氯甲基錫(Trimethyltin,TMT)是一種重要的職業(yè)危害毒物，也是一種重要的環(huán)境污染物。急性TMT暴露可引起多系統(tǒng)主要包括神經(jīng)系統(tǒng)的損害。TMT作用于中樞神經(jīng)系統(tǒng)，主要損傷作用機(jī)制為引起神經(jīng)細(xì)胞凋亡，引起神經(jīng)系統(tǒng)炎性反應(yīng)，對神經(jīng)組織造成氧化損傷等。TMT可以選擇性地誘導(dǎo)大腦邊緣系統(tǒng)，特別是海馬神經(jīng)元死亡，造成神經(jīng)系統(tǒng)損傷效應(yīng)。研究表明，BDNF在中樞神經(jīng)系統(tǒng)的主要作用是調(diào)節(jié)海馬神經(jīng)突觸可塑性。TMT暴露的大鼠大腦BDNF表達(dá)下調(diào)，而過表達(dá)BDNF的神經(jīng)元具有拮抗TMT神經(jīng)毒性的作用。但TMT對大腦神經(jīng)元的損傷機(jī)制特別是對神經(jīng)元樹突棘的損傷研究還不清楚，并且BDNF拮抗TMT損傷發(fā)揮神經(jīng)保護(hù)作用的機(jī)制尚待進(jìn)一步研究。因此，本課題選取TMT及BDNF為研究內(nèi)容，試圖理解TMT對神經(jīng)元的損傷作用機(jī)制及BDNF保護(hù)作用的分子機(jī)制，并為治療和預(yù)防TMT職業(yè)暴露造成的神經(jīng)元損傷提供思路。 突觸可塑性指突觸效率的功能性增強(qiáng)或降低，同時神經(jīng)信號傳遞強(qiáng)度的變化伴隨著神經(jīng)元突觸的結(jié)構(gòu)變化。并且，BDNF參與調(diào)節(jié)活動依賴的樹突棘發(fā)育及可塑性。近期研究表明BDNF可以作為成年腦中活動依賴性調(diào)節(jié)因子參與調(diào)控神經(jīng)結(jié)構(gòu)和功能變化。BDNF從樹突分泌出來后，立即與神經(jīng)營養(yǎng)因子受體TrkB結(jié)合，進(jìn)而促進(jìn)突觸前神經(jīng)遞質(zhì)的釋放并且增加突觸后蛋白的轉(zhuǎn)錄翻譯水平。Tau蛋白主要表達(dá)于神經(jīng)元樹突軸，獨(dú)立或與其他微管相關(guān)蛋白協(xié)同調(diào)節(jié)樹突軸微管功能。在神經(jīng)細(xì)胞中，tau蛋白與細(xì)胞膜相聯(lián)系或與微管發(fā)生相互作用。Tau蛋白表達(dá)或者結(jié)構(gòu)的變化可能影響其穩(wěn)定微管蛋白的功能。在生理?xiàng)l件下，tau蛋白可能在不同的位點(diǎn)發(fā)生磷酸化改變，進(jìn)而影響其穩(wěn)定微管的功能。成熟神經(jīng)元可以利用微管的動態(tài)性保持細(xì)胞體系的彈性，以適應(yīng)神經(jīng)網(wǎng)絡(luò)發(fā)生的各種變化。 方法： 第一部分TMT對神經(jīng)元毒性的觀察 以原代培養(yǎng)的胚胎期18天海馬神經(jīng)元為模型，用TMT處理后，用CCK-8的方法觀測BDNF對TMT的保護(hù)作用，觀察TMT處理后神經(jīng)元樹突棘形態(tài)的影響以及BDNF的保護(hù)作用，并明確TMT對神經(jīng)元樹突棘毒性的特征； 第二部分BDNF對神經(jīng)元突觸和樹突棘生長影響的觀察 以原代培養(yǎng)的胚胎期18天海馬神經(jīng)元為模型，用BDNF處理，觀察對原代培養(yǎng)海馬神經(jīng)元突觸生長和樹突棘形態(tài)的影響；利用質(zhì)粒轉(zhuǎn)染GFP或RFP標(biāo)記原代海馬神經(jīng)元樹突棘，細(xì)胞免疫熒光化學(xué)觀測tau蛋白和微管的共定位情況，突觸綜合體測量BDNF處理后突觸生長的變化，westernblot檢測tau蛋白及其磷酸化變化，明確BDNF處理海馬神經(jīng)元后，神經(jīng)元的形態(tài)突觸可塑性的變化以及tau蛋白所起的作用； 第三部分BDNF對Tau蛋白磷酸化及細(xì)胞分布的觀察 以RA分化的人神經(jīng)母細(xì)胞瘤SH-SY5Y和原代培養(yǎng)的胚胎期18天海馬神經(jīng)元為模型，用BDNF處理后，采用細(xì)胞免疫熒光化學(xué)的方法觀察tau蛋白的亞細(xì)胞分布情況，，Leica軟件測量SH-SY5Y細(xì)胞的突觸生長情況，westernblot檢測tau蛋白表達(dá)變化,明確tau蛋白亞細(xì)胞分布和BDNF處理后細(xì)胞的突觸生長的關(guān)系； 結(jié)果： 第一部分TMT引起樹突棘的異常形態(tài)改變 TMT處理體外培養(yǎng)14天的原代海馬神經(jīng)元24h，CCK-8檢測細(xì)胞活力顯著下降；加入BDNF與TMT共刺激后，CCK-8檢測細(xì)胞活力無顯著上升。原代海馬神經(jīng)元體外培養(yǎng)7天時轉(zhuǎn)染GFP質(zhì)粒，培養(yǎng)至20天，TMT和/或BDNF處理24h后固定細(xì)胞，共聚焦顯微鏡發(fā)現(xiàn)TMT處理后神經(jīng)元樹突棘出現(xiàn)異常圓形增大。加入BDNF處理后，樹突棘的異常圓形增大有減少的趨勢。BDNF與LiCl共刺激能夠同樣使神經(jīng)元樹突棘出現(xiàn)與TMT處理后高度相似的異常圓形增大。Westerbnlot實(shí)驗(yàn)結(jié)果表明，BDNF處理后可以同時在原代培養(yǎng)的海馬神經(jīng)元磷酸化Akt和ERK。而鋰單獨(dú)似乎不能影響Akt和ERK的磷酸化狀態(tài)。當(dāng)鋰與BDNF同時作用時，BDNF誘導(dǎo)的ERK的磷酸化沒有影響，但鋰卻抑制BDNF誘導(dǎo)的Akt磷酸化。TMT以及BDNF和LiCl聯(lián)合作用產(chǎn)生的神經(jīng)元樹突棘異常增大可能與ERK與PI3K-Akt之間的cross-talk平衡被破壞有關(guān)。 第二部分BDNF通過tau蛋白調(diào)節(jié)突觸和樹突棘生長 BDNF處理體外培養(yǎng)14天的海馬神經(jīng)元24h，Tau蛋白的表達(dá)都顯著升高，Ser262位點(diǎn)的磷酸化狀態(tài)在BDNF刺激后具有下降的趨勢。細(xì)胞免疫熒光化學(xué)實(shí)驗(yàn)也證明tau蛋白與微管蛋白在BDNF處理后的共定位得以增強(qiáng)。BDNF處理后tau蛋白的表達(dá)變化與樹突棘密度的增加一致。運(yùn)用shRNA技術(shù)在體外培養(yǎng)7天時下調(diào)tau蛋白表達(dá)水平后，培養(yǎng)至21天海馬神經(jīng)元樹突棘密度顯著下降。并且在shRNA下調(diào)tau蛋白的海馬神經(jīng)元中，24h BDNF刺激不能增加神經(jīng)元的樹突棘密度。 第三部分BDNF調(diào)節(jié)tau蛋白的磷酸化與亞細(xì)胞分布 通過免疫細(xì)胞化學(xué)方法，我們發(fā)現(xiàn)在未分化的缺乏神經(jīng)突起的SH-SY5Y細(xì)胞中，tau蛋白形成一個球體。相反，在維甲酸誘導(dǎo)分化5天的SH-SY5Y細(xì)胞中，tau蛋白分散分布在神經(jīng)細(xì)胞突起和胞體中。通過Western blot檢測方法，我們發(fā)現(xiàn)維甲酸的處理也增加了總tau蛋白水平而降低tau蛋白Ser262磷酸化水平。Tau蛋白表達(dá)上調(diào)和tau蛋白ser262磷酸化水平的下調(diào)與神經(jīng)細(xì)胞突起長度呈相關(guān)性(相關(guān)因子分別為r=0.94和r=-0.98)。當(dāng)原E18海馬神經(jīng)元用微管解聚劑nocodazole處理后，新生的神經(jīng)元突起丟失并且發(fā)生tau蛋白轉(zhuǎn)移到胞體的現(xiàn)象。這種遠(yuǎn)離突起的過程可以被BDNF一定程度上逆轉(zhuǎn)。 研究結(jié)論 基于以上研究結(jié)果，我們得出以下研究結(jié)論：TMT處理體外培養(yǎng)的海馬神經(jīng)元后發(fā)現(xiàn)神經(jīng)元的樹突棘出現(xiàn)異常的增大現(xiàn)象，可能是TMT的神經(jīng)毒性表現(xiàn)。BDNF具有保護(hù)TMT樹突棘損傷的潛力。TMT處理以及BDNF和LiCl聯(lián)合作用產(chǎn)生的神經(jīng)元樹突棘異常增大可能與ERK與Akt之間的cross-talk平衡被破壞有關(guān)。BDNF能夠調(diào)節(jié)原代海馬神經(jīng)元tau蛋白的表達(dá),這樣的調(diào)節(jié)具有劑量依賴關(guān)系和時效依賴關(guān)系。通過質(zhì)粒轉(zhuǎn)染原代培養(yǎng)海馬神經(jīng)元，下調(diào)tau蛋白表達(dá)，BDNF刺激神經(jīng)元樹突棘生長的現(xiàn)象被抑制，說明tau蛋白可能參與了BDNF調(diào)節(jié)神經(jīng)元突觸及樹突棘生長的信號通路。進(jìn)一步實(shí)驗(yàn)表明，BDNF調(diào)節(jié)樹突棘可塑性的作用可能是通過調(diào)節(jié)tau蛋白的表達(dá)量進(jìn)而影響tau蛋白穩(wěn)定微管能力來實(shí)現(xiàn)的；tau蛋白Ser262位點(diǎn)的磷酸化水平，總tau蛋白的表達(dá)量以及tau蛋白的亞細(xì)胞分布變化與神經(jīng)細(xì)胞突觸生長的顯著相關(guān)，tau蛋白的表達(dá)增加以及Ser262位點(diǎn)的去磷酸化可能以利于神經(jīng)細(xì)胞突觸的生長。本實(shí)驗(yàn)為TMT的神經(jīng)毒性特別是對海馬神經(jīng)元樹突棘的損傷提供新的證據(jù)，也揭示了BDNF可以通過tau蛋白調(diào)節(jié)突觸以及樹突棘生長發(fā)揮神經(jīng)保護(hù)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R114
本文編號：2138516
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R114
【共引文獻(xiàn)】
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