【作者】 罗鸣；

【导师】 伍钢；

【作者基本信息】 华中科技大学 ， 肿瘤学， 2011， 硕士

【摘要】 【目的】研究抗炎药物Corilagin抑制放射引起小胶质细胞(BV-2)炎症反应的作用及其防护的分子机制。【方法】细胞抑制实验(MTT)检测Corilagin对BV-2细胞抑制率;Corilagin预处理小胶质细胞,12 h后以0和32Gy 2个放射剂量照射BV-2细胞,Real-time PCR检测小胶质细胞照射后不同时间点炎症因子IL-1β、TNF-α表达水平;硝酸还原酶法检测细胞上清液中一氧化氮(NO)的含量;Western-blot检测各组细胞核转录因子NF-κB p65蛋白表达;激光共聚焦显微镜观察各组细胞标志物Iba-1的表达、NF-κB p65核转位及Nemo的表达。【结果】1~10μg/mL浓度范围内Corilagin对BV-2细胞增殖几乎无影响;照射后小胶质细胞表面标志物Iba-1表达明显,提示小胶质细胞激活,且炎症因子NO、TNF-α、IL-1β表达上调,而Corilagin(5μg/mL)可以显著抑制上述炎症因子的表达(tIL-1β=6.341,P<0.001;tTNF-α=3.411,P<0.007;tNO=3.134,P<0.015);照射后BV-2细胞内核转录因子NF-κB p65从胞浆转入胞核,免疫细胞化学进一步提示照射后细胞内活化蛋白Nemo表达增加,而IκB表达减少。Corilagin可抑制NF-κB活化蛋白Nemo,并显著抑制NF-κB p65的转位。【结论】Corilagin可能通过抑制途径照射后小胶质细胞NF-κB信号转导的活化,从而下调炎症因子表达,保护神经元。更多还原

【Abstract】 【Objective】The aim of this work was to explore the inhibitory effects of Corilagin on the production of pro-inflammatory cytokines in microglia stimulated by radiation.【Methods】The cytotoxicity of Corilagin was measured by MTT assay. Microglia BV-2 cells were stimulated by radiation(0, 32Gy)after pretreated with Corilagin for 12 hours. Realtime-PCR was used to detect the inflammatory cytokines such as IL-1β, TNF-αwhich were expressed by radiation-stimulated microglia BV cells on several time point. The production of Nitric Oxide (NO) was determined with nitrate reductase. The translocation of NF-κB p65 was measured by western-blot and immunocyto- chemical stain. Confocal microscopy was used to detected the expression of Iba-1, Nemo , IκB and the translocation of NF-κB p65.【Results】Corilagin showed no cytotoxicity for BV-2 cells within 1-10μg/mL. Irradiation was able to activate microglia cell with expression of Iba-1, and elevated the expression of inflammatory cytokines such as IL-1β, TNF-αand NO. Whereas, 5μg/mL Corilagin was significantly able to inhibit the production of IL-1β, TNF-αin activated microglia cells (tIL-1β=6.341,p<0.001;tTNF-α=3.411,p<0.007;tNO=3.134,p<0.015). Given a 32Gy of irradiation, NF-κB p65 in BV-2 translocate from cytoplasm to nuclear, correspondence with a increased Nemo and a inhibition of IκB, Corilagin significantly inhibited the expression of Nemo and the translocation and activation of NF-κB p65.【Conclusions】Corilagin could inhibit the activation of radiated microglia cells and down-regulate the expression of inflammatory cytokines, via inhibition of NF-κB signaling pathway.更多还原