细菌脂多糖增加小鼠耳蜗核因子κB的DNA结合活性

目的 探讨鼓室内注射细菌内毒素脂多糖（lipoplysaccharide,LPS）是否诱发小鼠耳蜗中转录因子核因子κB（nuclear factor-κB,NF－κB)的激活，建立耳蜗组织NF－κB激活的研究方法。方法 CBA小鼠12只，分别鼓室内注射LPS（6只）或生理盐水（6只），3h后耳蜗进行核蛋白提取并定量，将核提取物与^32P标记的NF－κB寡核苷酸探针进行结合，通过4．5％聚丙烯酰胺凝胶电泳分离结合与游离探针，放射自显影分析，以检测耳蜗中核蛋白提取物与特异性NF－κB探针的结合活性，并用NF－κB亚单位P65和P50抗体进行超滞后分析。结果 LPS组NF－κB的DNA结合活性较生理盐水组明显增高。超滞后分析表明预先与P65和P50抗体反应的LPS组出现明显滞后电泳带，提示NF－κB激活与P65和P50亚单位有关。结论 鼓室内注射LPS可诱发耳蜗NF－κB激活，该方法可为进一步研究耳蜗NF－κB激活提供阳性动物模型。

Lipopolysaccharide enhances DNA binding activity of nuclear factor-kappa B in the mouse cochlea]

OBJECTIVE: To ascertain whether inoculation of lipopolysaccharide (LPS) into the mouse cochlea might induce enhancement of DNA binding activity of NK-kappa B and to establish a positive model of NF-kappa B activation in the cochlea. METHODS: Two groups of CBA mice were transtympanically injected either with 10 micrograms of LPS or an equal volume of saline. The nuclear proteins of the cochlea were extracted, quantitated and bound with 32P-radiolabeled oligonucleotide probe for NF-kappa B. The bound and free probes were separated by electrophoresis on a 4.5% native polyacrylamide gel. Supershift assays were carried out using the antibodies against NF-kappa B subunit P65 and P50. RESULTS: The radiolabeled probe bound differently to nuclear extracts from the LPS-treated and the saline-treated mouse cochleae, showing that LPS inoculation significantly increased NF-kappa B binding activity. Supershift analysis demonstrated that the enhancement of DNA binding activity was related to both P65 and P50 subunits. CONCLUSION: LPS could induce NF-kappa B activation in the cochlea. Moreover, this study implies that tissues from LPS-inoculated cochlea might serve as a useful positive control in the study on activation of NF-kappa B in cochlea.