激光扫描共聚焦显微镜检测核因子κB

Activation of nuclear factor κB assayed by laser scanning confocal microscope

目的 探讨激光扫描共聚焦显微镜 (CLSM )检测核因子κB(NF κB)的方法。方法 用肿瘤坏死因子α(TNF α)刺激PC1 2细胞NF κB活化 ,间接免疫荧光 (FITC)标记代表NF κB的P6 5 亚基 ,碘化丙啶 (PI)标记细胞DNA指示核区 ,用CLSM对NF κB进行定位显示和定量分析 ,以WesternBlot检测结果作为对照标准。结果 TNF α刺激后PC1 2细胞经CLSM显示 :核区内出现代表NF κB的绿色荧光 ,其核区与胞浆区比值 (FN/FC)明显大于未经TNF α刺激的PC1 2细胞 ;而未经TNF α刺激的PC1 2细胞红色的核区内未出现绿色荧光。WesternBlot显示TNF α刺激后PC1 2细胞核提取物呈阳性反应 ,即出现代表NF κB/P6 5 蛋白的条带 ,未经TNF α刺激的PC1 2细胞核提取物呈阴性反应 ,即核提取物中未出现代表NF κB/p6 5 蛋白的条带 ,CLSM检测NF κB与WesternBlot检测结果一致。结论 CLSM检测NF κB既可定位显示 ,又可定量分析 ,能显示单个细胞的NF κB状态 ,且具有同时显示细胞形态等优点 ,是研究细胞核因子的有效方法。

Objective To investigate the method of detecting nuclear factor κB(NF κB) by laser scanning confocal microscope(CLSM).Methods Using two kinds of techniques: Western Blot and CLSM to detect the activation of NF κB in PC12 cell stimulated by TNF α with FITC tagged antibody and its presence in the nucleus via a via cytoplasm was monitored by measuring the green fluorescence integrated over the nucleus, which was counterstained with propidium iodide(PI),respectively.Results NF κB/P 65 associated fluorescence can be seen in the nuclei of TNF α stimulated PC12 cells,no such fluorescence were seen in the nuclei of no stimulated PC12 cells.The protein bands stand for nuclear NF κB/P 65 from stimulated PC12 cells nuclear extracts were detected on nitrocellulose. As for unstimulated PC12 cells,the protein bands were not detected.Conclusion The activation of NF κB may be detected by CLSM not only orientationally but also quantitively.The described method can show the state of NF κB in a single cell with its morphology,and has a potential to be used as a functional assay to monitor intracellular translocation of other transcriptional activators.