CLSM检测局灶性脑缺血NFκB的转录活性.docVIP

CLSM检测局灶性脑缺血NF-κB的转录活性 　　作者：毛诗贤， 杨期东 作者单位：贵阳医学院附院 神经内科， 贵州 贵阳 550004 　　【摘要】 目的： 探讨共聚焦激光扫描显微镜(confocal laser scanning microscope,CLSM)结合荧光双标技术检测局灶性脑缺血nuclear factor-kappa B(NF-ΚB)转录活性的方法。方法： 线栓法制作大鼠大脑中动脉缺血2 h再灌注模型，给予clasto-lactacystin Β-lactone抑制NF-ΚB的转录激活，制作石蜡切片，利用CLSM结合Fluoresceinisothiocyanate(FITC)标记NF-ΚB p65亚基和Propidium Iodide(PI)标记细胞核的荧光双标技术对NF-ΚB进行定位和定量分析。结果： 脑缺血再灌注后， NF-ΚB激活，CLSM显示细胞核中出现代表NF-ΚB的绿色荧光，NF-ΚB的转录活性被抑制后，细胞核中的绿色荧光减少，细胞核区绿色荧光与细胞浆区绿色荧光的比值减少。结论： CLSM结合荧光双标技术是定位、定量分析NF-ΚB转录活性的良好方法。 　　【关键词】 脑缺血; 再灌注损伤; 显微镜检查，聚焦; 大鼠，Sprague-Dawley; 共聚焦激光扫描显微镜; 荧光双标技术; 核因子 　　Transcriptional Activity of NF-ΚB Detected by Confocal Laser Scanning 　　Microscope after Focal Cerebral Ischemia and Reperfusion 　　MAO Shixian1, YANG Qidong2 　　(1. Department of Neurology, The Affiliated Hospital of Guiyang Medical College, Guiyang 550004, Guizhou, China; 　　2.Neurology Department, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China) 　　[Abstract] Objective: To investigate the method of detecting nuclear factor-kappa B (NF-ΚB) transcriptional activity by using confocal laser scanning microscope (CLSM) combined with fluorescence dual-labeling technique in a rat model of focal cerebral ischemia and reperfusion. Methods: Transient focal ischemia and reperfusion was produced by middle cerebral artery occlusion for 2 hours in Sprague-Dawley rats. The activation of NF-ΚB was inhibited by clasto-lactacystin Β-lactone. Paraffin section was made. Quantification and location of NF-ΚB was analyzed by CLSM combined with fluorescence dual-labeling technique, in which NF-ΚB p65 subunit was marked with fluoresce in isothiocyanate (FITC) and nuclei were marked using propidium iodide (PI).Results: NF-ΚB was activated after focal ischemia and reperfusion. NF-ΚB associated green fluorescence was seen in the nuclei with CLSM. When NF-ΚB was inactivated, the green fluorescence in the nuclei decreased, and the ratio of green fluorescence in the nuclei and