本文選題：SKF83959　切入點(diǎn)：小膠質(zhì)細(xì)胞　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究sigma-1受體別構(gòu)調(diào)節(jié)劑SKF83959對脂多糖(lipopolysaccharide,LPS)介導(dǎo)的小膠質(zhì)細(xì)胞炎癥反應(yīng)的抗炎作用以及炎癥介質(zhì)釋放所引起的神經(jīng)毒性的保護(hù)作用,并進(jìn)一步探討SKF83959抗炎作用的分子機(jī)制。方法:利用LPS刺激的BV2小膠質(zhì)細(xì)胞,MTT法檢測細(xì)胞活性;收集BV2細(xì)胞培養(yǎng)液上清,分別用Griess法和ELISA法檢測一氧化氮(NO)和腫瘤壞死因子(TNF-α)的含量;收集BV2細(xì)胞超聲破碎后進(jìn)行蛋白定量,用ELISA法檢測細(xì)胞內(nèi)DHEA的含量;Quantitative real-time PCR(Q-PCR)方法檢測炎癥介質(zhì)TNF-α、IL-1β和iNOS的mRNA表達(dá)水平;Western Blot方法檢測iNOS蛋白的表達(dá)以及MAPKs和IKK/IκBα蛋白磷酸化水平;收集細(xì)胞,流式細(xì)胞術(shù)檢測細(xì)胞內(nèi)活性氧(ROS)的水平;用放射性配基結(jié)合實(shí)驗(yàn)測定SKF83959對脫氫表雄酮(dehydroepiandrosterone,DHEA)與sigma-1受體結(jié)合能力的影響;BV2小膠質(zhì)細(xì)胞條件培養(yǎng)基與HT-22海馬神經(jīng)元細(xì)胞共培養(yǎng),收集細(xì)胞,采用流式細(xì)胞術(shù)檢測神經(jīng)元細(xì)胞凋亡水平,研究SKF83959對LPS介導(dǎo)的BV2小膠質(zhì)細(xì)胞炎癥介質(zhì)引起的神經(jīng)毒性的保護(hù)作用。結(jié)果:SKF83959顯著抑制LPS激活的BV2小膠質(zhì)細(xì)胞炎癥反應(yīng),但被sigma-1受體拮抗劑BD1047和BD1063以及DHEA合成抑制劑酮康唑阻斷;SKF83959促進(jìn)了DHEA與sigma-1受體結(jié)合,并以正向協(xié)同的方式增強(qiáng)了外源性DHEA的抗炎作用;Sigma-1受體拮抗劑BD1047和BD1063也阻斷了SKF83959和DHEA的協(xié)同抗炎作用;在小膠質(zhì)細(xì)胞條件培養(yǎng)實(shí)驗(yàn)中,SKF83959可以改善小膠質(zhì)細(xì)胞炎癥介質(zhì)對HT-22神經(jīng)元細(xì)胞的神經(jīng)毒性;SKF83959的抗炎作用與MAPK/ERK和NFκB信號通路激活以及D1受體活化并不相關(guān)。結(jié)論:SKF83959通過別構(gòu)調(diào)節(jié)sigma-1受體抑制BV2小膠質(zhì)細(xì)胞的炎癥反應(yīng)并對激活的小膠質(zhì)細(xì)胞的炎癥介質(zhì)所引起的神經(jīng)毒性具有保護(hù)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- 偽 (TNF- 偽) 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- 偽, 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 偽 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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R741

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本文編號：1577574