丙烯酰胺致NB-1细胞突触损伤与SNARE效应相关性研究

发布时间：2018-03-30 13:31

本文选题：丙烯酰胺　切入点：NB-细胞　出处：《毒理学杂志》2015年02期

【摘要】：目的构建人神经母细胞瘤NB-1细胞神经元突触损伤的细胞模型,探讨丙烯酰胺(acrylamide,ACR)对神经突触损伤的机制。方法将NB-1细胞通过二丁酰环腺苷酸(db-c AMP)诱导为成熟神经元后,MTT检测ACR不同剂量(0、25、50、100、150、200和250μg/ml)和不同染毒时间(24、48和72 h)对NB-1细胞相对存活率的影响;免疫印记(western blot)技术检测神经元突触损伤前后突触素I(Synapsin I)磷酸化和非磷酸化蛋白表达变化,光学显微镜和电子显微镜记录神经元突触损伤前后形态、结构变化,免疫共沉淀试验确定神经元突触损伤前后N-甲基马来酰胺敏感因子(NSF)附着蛋白受体(SNARE)的结合/解离的变化。结果根据MTT试验结果确定ACR染毒剂量为0、25、50和100μg/ml,染毒时间为48 h;随着ACR染毒剂量的增加Synapsin I磷酸化和非磷酸化蛋白表达降低;在25μg/ml ACR染毒剂量下可见兴奋性和抑制性神经递质,而在50μg/ml染毒剂量下可见抑制性递质;随ACR染毒剂量的增加SNARE复合体中突触融合蛋白(syntaxin)和突触小体相关蛋白(SNAP-25)呈解离状态。结论 ACR对NB-1细胞突触损伤后,SNARE复合体中syntaxin和SNAP-25结合状态与Synapsin I、P-Synapsin I蛋白表达降低呈正相关,与P-Synapsin I/Synapsin I差异性表达呈负相关;突触内特异性神经递质的传递和释放可能与P-Synapsin I/Synapsin I差异性表达所调控的SNARE复合体的解离状态导致的突触功能损伤相关。
[Abstract]:Objective to construct a cell model of synaptic injury in NB-1 cells of human neuroblastoma. To investigate the mechanism of synaptic injury induced by acrylamide acrylamide acrylamide (ACR). Methods NB-1 cells were induced into mature neurons by dibutyryl cyclic adenylate db-c AMP.Methods NB-1 cells were treated with different doses of ACR at different doses (0 25, 50 100150 200 and 250 渭 g / ml) and at different exposure times (24 48 and 72 h). The effect of relative survival rate; The expression of synaptophysin I (I(Synapsin I) phosphorylated and non-phosphorylated protein in neurons before and after synaptic injury were detected by Western blot technique. The morphological and structural changes of synaptic neurons before and after synaptic injury were recorded by optical microscope and electron microscope. The immunoprecipitation assay was used to determine the binding / dissociation of NMSF-NSFR receptor before and after synaptic injury. Results according to the results of MTT test, the dose of ACR was determined to be 0 ~ 2550 渭 g / ml and 100 渭 g / ml, respectively. The expression of Synapsin I phosphorylation and non-phosphorylated protein decreased with the increase of the dose of ACR. Excitatory and inhibitory neurotransmitters were observed at the dose of 25 渭 g/ml ACR and inhibitory neurotransmitters at the dose of 50 渭 g/ml. The synaptic fusion protein syntaxin (syntaxin) and synaptosome associated protein (SNAP-25) in the SNARE complex were dissociated with the increase of the dose of ACR. Conclusion there is a positive correlation between the expression of syntaxin and SNAP-25 in the SNARE complex after synaptic injury of NB-1 cells induced by ACR and the decrease of the expression of Synapsin Iron-Synapsin I protein. The transmission and release of specific neurotransmitters in the synapses may be related to the synaptic function damage caused by the dissociation of the SNARE complex regulated by the differential expression of P-Synapsin I/Synapsin I.
【作者单位】：河北联合大学公共卫生学院;中国疾病预防控制中心职业卫生与中毒控制所;中国疾病预防控制中心职业卫生与中毒控制所化学安全与健康重点实验室;
【基金】：国家自然科学基金(81273110) 公益性行业科研专项(201402021) 国家科技支撑计划(2014BAI12B00)
【分类号】：R114

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