贵州医科大学学报

2008, No.130(01) 49-51+54

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CLSM检测局灶性脑缺血NF-κB的转录活性
Transcriptional Activity of NF-κB Detected by Confocal Laser Scanning Microscope after Focal Cerebral Ischemia and Reperfusion

毛诗贤;杨期东;
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)

摘要(Abstract):

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2h再灌注模型,给予clasto-lactacystinβ-lactone抑制NF-κB的转录激活,制作石蜡切片,利用CLSM结合Fluorescein-isothiocyanate(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。
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 cytoplasm reduced.Conclusion: CLSM combined with fluorescence double labeling technique is a good method for location and quantitative analysis of NF-κB.

关键词(KeyWords): 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
brain ischemia; reperfusion injury; microscopy,confocal; rats,Sprague-Dawley; confocal laser scanning microscope; fluorescence dual-labeling technique; nuclear factor-kappa B

Abstract:

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基金项目(Foundation):

作者(Author): 毛诗贤;杨期东;
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)

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DOI: 10.19367/j.cnki.1000-2707.2008.01.018

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