别构调节sigma-1受体在小胶质细胞介导炎症中的作用及其机制研究

发布时间：2018-03-07 02:31

本文选题：SKF83959　切入点：小胶质细胞　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:研究sigma-1受体别构调节剂SKF83959对脂多糖(lipopolysaccharide,LPS)介导的小胶质细胞炎症反应的抗炎作用以及炎症介质释放所引起的神经毒性的保护作用,并进一步探讨SKF83959抗炎作用的分子机制。方法:利用LPS刺激的BV2小胶质细胞,MTT法检测细胞活性;收集BV2细胞培养液上清,分别用Griess法和ELISA法检测一氧化氮(NO)和肿瘤坏死因子(TNF-α)的含量;收集BV2细胞超声破碎后进行蛋白定量,用ELISA法检测细胞内DHEA的含量;Quantitative real-time PCR(Q-PCR)方法检测炎症介质TNF-α、IL-1β和iNOS的mRNA表达水平;Western Blot方法检测iNOS蛋白的表达以及MAPKs和IKK/IκBα蛋白磷酸化水平;收集细胞,流式细胞术检测细胞内活性氧(ROS)的水平;用放射性配基结合实验测定SKF83959对脱氢表雄酮(dehydroepiandrosterone,DHEA)与sigma-1受体结合能力的影响;BV2小胶质细胞条件培养基与HT-22海马神经元细胞共培养,收集细胞,采用流式细胞术检测神经元细胞凋亡水平,研究SKF83959对LPS介导的BV2小胶质细胞炎症介质引起的神经毒性的保护作用。结果:SKF83959显著抑制LPS激活的BV2小胶质细胞炎症反应,但被sigma-1受体拮抗剂BD1047和BD1063以及DHEA合成抑制剂酮康唑阻断;SKF83959促进了DHEA与sigma-1受体结合,并以正向协同的方式增强了外源性DHEA的抗炎作用;Sigma-1受体拮抗剂BD1047和BD1063也阻断了SKF83959和DHEA的协同抗炎作用;在小胶质细胞条件培养实验中,SKF83959可以改善小胶质细胞炎症介质对HT-22神经元细胞的神经毒性;SKF83959的抗炎作用与MAPK/ERK和NFκB信号通路激活以及D1受体活化并不相关。结论:SKF83959通过别构调节sigma-1受体抑制BV2小胶质细胞的炎症反应并对激活的小胶质细胞的炎症介质所引起的神经毒性具有保护作用。
[Abstract]:Aim: to study the anti-inflammatory effect of sigma-1 receptor allosteric modulator (SKF83959) on lipopolysaccharide (LPS) -mediated inflammation of microglia and the neurotoxicity induced by the release of inflammatory mediators. Methods: BV2 microglia stimulated by LPS were used to detect the cell activity, and the supernatant of BV2 cell culture medium was collected. The contents of nitric oxide (no) and tumor necrosis factor TNF- 伪 (TNF- 伪) were detected by Griess and ELISA, respectively. Quantitative real-time PCR Q-PCR method was used to detect the expression of mRNA in inflammatory mediators TNF- 伪, IL-1 尾 and iNOS. Western Blot was used to detect the expression of iNOS protein and the phosphorylation of MAPKs and IKK/I 魏 B 伪 protein. The effect of SKF83959 on the binding ability of dehydroepiandrosterone (Dehydroepiandrosterone) to sigma-1 receptor was determined by flow cytometry, and the co-culture of HT-22 hippocampal neurons with BV2 microglial conditioned medium. The apoptotic level of neurons was detected by flow cytometry, and the protective effect of SKF83959 on the neurotoxicity induced by BV2 microglial inflammatory mediators mediated by LPS was studied. Results: SKF83959 significantly inhibited the inflammatory response of BV2 microglia activated by LPS. However, SKF83959 was blocked by BD1047 and BD1063, a sigma-1 receptor antagonist, and ketoconazole, an inhibitor of DHEA synthesis, which promoted the binding of DHEA to sigma-1 receptor. The anti-inflammatory effect of exogenous DHEA was enhanced by positive synergism. BD1047 and BD1063 also blocked the synergistic anti-inflammatory effects of SKF83959 and DHEA. In microglial conditioned culture experiment, SKF83959 can improve the neurotoxicity of microglial inflammatory mediators on HT-22 neurons. The anti-inflammatory effect of SKF83959 is not related to the activation of MAPK/ERK and NF 魏 B signaling pathway and the activation of D1 receptor. Conclusion\% SKF83959 is not related to the activation of MAPK/ERK and NF 魏 B signaling pathway. Allogeneic regulation of sigma-1 receptor inhibits the inflammatory response of BV2 microglia and protects against neurotoxicity induced by inflammatory mediators of activated microglia.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R741

【相似文献】