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背景:近年来对于缺血半影区的研究,涉及脑组织血流量、能量代谢、蛋白质合成率、细胞凋亡以及相关蛋白质表达等多个方面,然而对于DNA单链与双链损伤的动态分布研究很少。目的:研究大鼠大脑中动脉缺血再灌注时,DNA损伤随再灌注时程在各脑区的动态分布情况。设计:随机对照的实验研究。地点和对象:本实验在华中科技大学同济医学院附属同济医院完成;选择健康纯系Wistar大鼠28只,体质量200～230g,雌雄各半,所有动物均由同济医学院动物饲养中心提供。干预:用线栓闭合大鼠大脑中动脉30min,然后分别再灌注30min,1,2,4,6,12,24,48h。采用原位PANT(DNA聚合酶I介导的生物素标记的dATP缺口平移)及原位TUNEL(末端脱氧核苷酸转移酶介导的dUTP末端标记),分别检测DNA损伤的单链断裂及双链断裂。主要观察指标:观察单链断裂细胞和双链断裂细胞在大鼠前囟水平冠状切面的脑组织切片各区域的分布。结果:缺血30min(再灌注以前)各脑区均未检测到PANT或TUNEL阳性细胞。再灌注1h在尾壳核区检测到DNA单链断裂,再灌注2h在该区和梨状皮质检测到DNA双链断裂;再灌注24h以前各时间点DNA单链断裂和双链断裂细胞数依次增多,并且出现在顶叶皮质和额叶皮质,再灌注48h二者均减少;再灌注各时间点,分布在尾壳核和梨状皮质的PANT或TUNEL阳性细胞
BACKGROUND: In recent years, studies on the ischemic penumbra involve many aspects such as cerebral blood flow, energy metabolism, protein synthesis, apoptosis and related protein expression. However, the dynamic distribution of single and double strand DNA damage Little research. OBJECTIVE: To study the dynamic changes of DNA damage during reperfusion in different brain regions of rat middle cerebral artery during ischemia-reperfusion. Design: Randomized controlled experimental study. Location and Subject: The experiment was performed at Tongji Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology. Totally 28 healthy Wistar rats were selected and their body weight was 200-230g. All animals were provided by Animal Rehabilitation Center of Tongji Medical College. Intervention: The middle cerebral artery of rats were closed for 30 minutes with a thrombus and then reperfused for 30 min, 1, 2, 4, 6, 12, 24, 48 h respectively. In situ PANT (DNA polymerase I-mediated biotinylated dATP nick translation) and in situ TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP end labeling) were used to detect single-stranded DNA breaks and double Chain break. MAIN OUTCOME MEASURES: The distribution of single-stranded and double-stranded cleaved cells in each section of the coronal section of the rat bregma level was observed. Results: PANT or TUNEL positive cells were not detected in all brain regions at 30 min after ischemia (before reperfusion). DNA single strand breaks were detected in the caudate putamen 1 h after reperfusion, and DNA double strand breaks were detected in this area and piriform cortex 2 h after reperfusion. The number of single-stranded DNA breaks and double-stranded breaks at each time point before reperfusion 24h Increased, and appeared in the parietal cortex and frontal cortex, 48h after reperfusion both reduced; reperfusion at various time points, located in the caudate putamen and piriform cortex PANT or TUNEL-positive cells