豚鼠椎基底动脉缺血/再灌注耳蜗损伤NF-кBp65表达的研究

Changes of NF-κBp65 following vertebrobasilar artery ischemia reperfusion in guinea pigs

目的探讨豚鼠椎基底动脉缺血/再灌注时是否存在NF-кBp65的激活。方法经颅底径路建立豚鼠椎基底动脉缺血/再灌注耳蜗损伤模型,采用石蜡包埋免疫组织化学技术,对各组动物近中轴位切片行NF-кBp65免疫组织化学染色,光学显微镜下观察在耳蜗组织中的表达情况;提取缺血再灌注侧耳蜗组织的细胞核和细胞浆蛋白,应用电泳迁移率滞后分析检测细胞核内NF-кBDNA结合活性。结果正常豚鼠耳蜗螺旋神经节、Corti's器、血管纹、螺旋韧带的细胞浆和细胞核内NF-кBp65染色呈阴性,缺血再灌注12h可见明显表达,24h表达最强,48h表达逐渐减弱;正常耳蜗组织中可见少量的NF-кB活化,在缺血再灌注的耳蜗组织中NF-кBDNA的结合活性显著上调,再灌注24h的结合活性最高,再灌注48h后NF-кBDNA的结合活性下降。结论耳蜗缺血再灌注损伤时,NF-κBp65在耳蜗出现异常表达,NF-κBDNA结合活性增高。

[Objective] To investigate the activation of the nuclear factor-kappaBp65 in the cochlea of guinea pigs induced by vertebrobasilar artery ischemia reperfusion. [Method] The models of vertebrobasilar artery ischemia reperfusion were established via the skull base approach. The spatial and temporal features of NF-κBp65 induction following ischemia reperfusion were studied by paraffin embedded immunohistochemistry. The nuclear protein were exlxacted from the cochlea tissue. Electrophoretic mobility shift assay （EMSA） were used to detect the nuclear protein DNA binding activity of NF-κ B. [Results] The negative reaction for NF-κ Bp65 staining was found in the cochlea in normol control group. The positive reaction was found in the cytoplasm and/or nucleus of the Corti, spiral ganglion cells （SGCs）, spiral ligament and stria vasucularis in the all iscbemia reperfusion groups. EMSA showed that ischemia-reperfusion injury significantly increased the DNA binding activity ofNF-κB. [Conclusion] The obvious upregulation ofNF-κ B activation is observed in ischemia-reperfusion cochlea tissue, as compared to the normal control group. NF-κBp65 may be involved in the ischemia reperfusion injury.