【作者】 吴冰珊；

【导师】 苏东辉； 朱玲；

【作者基本信息】 福建医科大学 ， 分子免疫学， 2003， 硕士

【摘要】 目的 采用脂多糖(LPS)和β-淀粉样蛋白分别刺激小鼠小胶质细胞(N9)，测定APP、FPR2、TNF-α和IL-1β的表达，同时研究新型趋化因子Fractalkine(FKN)对这些基因表达的调制作用，探讨Fractalkine在抗早老性痴呆发病机制中对神经退行性病变的效应。 方法 对数生长期的N9细胞分别在LPS、Aβ、FKN、LPS+FKN、Aβ+FKN干预下孵育24h。在不同时间点收集贴壁细胞，以RT-PCR法检测细胞因子/受体，吸取细胞培养上清以ELISA法定量检测TNF-α。 结果 LPS干预体外培养的N9细胞，使APP、FPR2的表达升高，炎症因子TNF-α，和IL-1β的表达达峰后在24h明显降低。以Aβ代替LPS激活N9细胞，同样可检测到APP、TNF-α和IL-1β的表达。N9细胞预先以重组FKN孵育后施加Aβ刺激，APP、IL-1βmRNA和TNF-α(RT-PCR或ELISA法检测)的表达较单独Aβ作用者明显下降，而FPR2mRNA表达则显著升高。 结论 研究发现与Alzheimer’s病紧密相关的Aβ蛋白同LPS一样可激活小胶质细胞表达APP、TNF-α和IL-1β。有意义的是外源性Fractalkine可能在CNS抑制β-淀粉样蛋白及炎症因子的产生中发挥重要作用。更多还原

【Abstract】 Objective The present study was to investigate the expression of the genes encoding APP, FPR2, TNF-a and IL-1β in a murine microglia cell line N9 stimulated with bacterial endotoxin and beta-amyloid protein, respectively, and to study the effect of fractalkine, a new kind of chemokine, on the modulation of the cytokine/receptor expression in these experimental steps. This investigation is helpful to the exploration of the role of fractalkine in the mechanisms of neuro-degeneration in Alzheimer’s disease.Methods Goups of log-phase N9 cells were cultured for 24h in the presence of LPS, beta-amyloid protein, fractalkine, LPS plus fractalkine, beta-amyloid protein plus fractalkine, respectively. Culture cells were harvested at different time points of incubation for the detection of cytokine/receptor mRNA expression by means of RT-PCR and culture supernatants were assayed for TNF-a production by ELISA.Results Incubation of N9 cells with LPS resulted in a successive increase of FPR2 expression but a low level expression of APP mRNA throughout 24h with a significant decline at 4h. The expression of inflammatory cytokines, IL-1β and TNF-a, had a peak time and significantly decreased at 24h. When Aβ was added to cultures instead, the expression of APP, IL-1β and TNF-a were also detected. When recombinant fractalkine was pre-incubated with N9 cells, the expression of APP and IL-lp mRNA and TNF-a (either detected by RT-PCR or ELISA) induced by Ap was significantly decreased while FPR2 mRNA expression was enhanced.Conclusion These findings suggest that beta-amyloid protein, similar to LPS, can stimulate the expression of TNF-a, IL-1β and Aβ precursor mRNA, which has been thought to be associated with the onset of Alzheimer’s disease. Interestingly, it is indicated that exogenous FKN might be able to play an inhibitory role on the Ap and inflammatory cytokine production in CNS.更多还原