食源性铅暴露对发育期原代海马神经元元突触传递的影响及可能机理

发布时间：2018-01-29 18:12

本文关键词： 铅突触传递 H3K27me3 synapsin1 CDK5　出处：《合肥工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景和目的:食源性铅中毒事件越来越受到人们的广泛关注。大量的研究表明,铅可导致大脑的神经系统的损伤,进而影响学习记忆和认知能力,其中对发育期儿童的损伤尤为明显。突触神经递质的传递是维持大脑神经活动正常工作的基础,但是铅对发育期的神经递质的传递的影响及其机理尚不明确。因此,本课题旨在研究“慢性铅暴露对原代海马神经元突触传递的影响及可能机理研究”。方法:1.以Sprague-Dawley(SD)新生大鼠(出生24h以内)为模型,取海马CA1组织进行离体培养,在第三天时加入5μM的醋酸铅进行慢性铅暴露处理。在第14天时,利用电生理膜片钳技术,记录突触后微小电流(mEPSC和mIPSC),以评价慢性铅暴露对兴奋性突触传递和抑制性突触传递的影响。2.整体动物实验以SD母鼠为研究模型,250 ppm醋酸铅在SD大鼠孕期进行暴露至幼鼠出生30天。利用透射电镜观察CA1组织突触前膜囊泡分布;Q-PCR检测SNARE单体syntaxin1a、SNAP-25、VAMP2的m RNA表达量;Western Blot检测了SNARE复合体的蛋白表达量;电生理记录PPR。另一方面,为了阐明慢性铅暴露对突触传递影响的可能机理,以离体培养的原代海马神经元为模型,5μM的铅暴露3至14天。1.用Western Blot检测H3K27me3的蛋白含量,以及synapsin1和synapsin1的磷酸化水平。2.染色质免疫共沉淀技术检测检测H3K27me3与synapsin1的互作能力。3.Q-PCR检测H3K27me3下游调控基因synapsin1、synapsin2、synaptotagmin6以及CDK5的m RNA的表达量。4.电生理膜片钳技术进一步验证慢性铅暴露导致的递质释放的抑制是否有CDK5的参与。结果:(1)慢性铅暴露显著降低了原代海马神经元m EPSC和m IPSC的频率,但是对m EPSC和m IPSC的幅度无显著变化。(2)透射电镜的结果显示慢性铅暴露使突触前膜的囊泡在突触前膜的分布更加的弥散;Q-PCR结果表明慢性铅暴露导致了SNAP-25和VAMP2的m RNA显著下降,syntaxin1a的m RNA的含量显著上升;但是Western Blot的结果显示,SNARE复合体的蛋白含量不变。电生理结果显示慢性铅暴露使得囊泡的释放能力降低。(3)慢性铅暴露导致H3K27me3的蛋白含量显著下降,通过基因芯片技术筛选出与囊泡释放相关的基因,Q-PCR结果显示慢性铅暴露显著降低了synapsin1的m RNA水平,但synapsin2的水平显著上升。Chip实验结果显示慢性铅暴露组中H3K27me3与synapsin1的结合能力上升。(4)慢性铅暴露显著降低了synapsin1的第553位色氨酸位点的磷酸化水平,对synapsin1的总蛋白含量没有变化。慢性铅暴露显著提高了synapsin1磷酸化位点的上游CDK5的m RNA的表达量。结论:(1)慢性铅暴露抑制了原代海马神经元兴奋性和抑制性神经递质的释放,但是没有改变突触后膜的电流强度。(2)慢性铅暴露改变了突触前膜囊泡的分布,并且抑制了突触前膜囊泡的释放能力。(3)慢性铅暴露通过提高CDK5的表达量导致synapsin1的第553位色氨酸位点的磷酸化进而抑制神经递质的释放。
[Abstract]:Background and objective: people pay more and more attention to foodborne lead poisoning. A large number of studies have shown that lead can lead to the damage of the nervous system of the brain, thus affecting learning, memory and cognitive ability. The transmission of synaptic neurotransmitters is the basis of maintaining the normal work of brain nerve activity. However, the effect of lead on the transmission of neurotransmitters at the developmental stage and its mechanism are not clear. The purpose of this study was to study the effect of chronic lead exposure on synaptic transmission in primary hippocampal neurons and its possible mechanism. Within 24 hours of birth. The hippocampal CA1 tissue was cultured in vitro and treated with 5 渭 M lead acetate on the third day. On the 14th day, electrophysiological patch clamp technique was used. To evaluate the effects of chronic lead exposure on excitatory synaptic transmission and inhibitory synaptic transmission. 250 ppm lead acetate was exposed to SD rats during pregnancy to 30 days after birth. The distribution of presynaptic vesicles in CA1 tissues was observed by transmission electron microscope. Q-PCR was used to detect the m RNA expression of SNARE monomer SNAP-25 VAMP2. The protein expression of SNARE complex was detected by Western Blot. On the other hand, in order to elucidate the possible mechanism of the effect of chronic lead exposure on synaptic transmission, the primary hippocampal neurons in vitro were used as the model. The protein content of H3K27me3 was detected by Western Blot. The phosphorylation level of synapsin1 and synapsin1. 2. Chromatin immunoprecipitation assay was used to detect the interaction between H3K27me3 and synapsin1. Q-PCR was used to detect the downstream regulation gene synapsin1 of H3K27me3. Synapsin2. Synaptotagmin6 and CDK5 m. RNA expression. 4. Electrophysiological patch clamp technique to further verify whether CDK5 is involved in the inhibition of transmitter release induced by chronic lead exposure. 1) chronic lead exposure significantly reduced the frequency of m EPSC and m IPSC in primary hippocampal neurons. But the amplitude of m EPSC and m IPSC did not change significantly. The results of transmission electron microscope showed that chronic lead exposure made the vesicles of presynaptic membrane more diffuse in the presynaptic membrane. Q-PCR results showed that chronic lead exposure resulted in a significant decrease of m RNA in SNAP-25 and VAMP2. The content of m RNA in syntaxin 1a was significantly increased. But the result of Western Blot shows. The electrophysiological results showed that chronic lead exposure decreased the release ability of vesicles.) chronic lead exposure resulted in a significant decrease in the protein content of H3K27me3. The results of Q-PCR showed that chronic lead exposure significantly reduced the level of m RNA in synapsin1. But the level of synapsin2 increased significantly. The results of Chip experiment showed that the binding ability of H3K27me3 to synapsin1 in chronic lead exposure group was increased. Chronic lead exposure significantly reduced the phosphorylation level of tryptophan site 553 in synapsin1. Chronic lead exposure significantly increased the expression of m RNA in CDK5 upstream of synapsin1 phosphorylation site. Chronic lead exposure inhibited the release of excitatory and inhibitory neurotransmitters from primary hippocampal neurons. However, chronic lead exposure did not change the current intensity of postsynaptic membranes.) chronic lead exposure changed the distribution of presynaptic vesicles. And inhibited the release ability of presynaptic vesicles. Chronic lead exposure inhibits the release of neurotransmitters by increasing the expression of CDK5 resulting in phosphorylation of the 553rd tryptophan site in synapsin1.
【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R595.2

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