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目的:研究末端脱氧核苷酸转移酶介导的dUTP缺口末端标记(TUNEL)、DNA聚合酶I介导的生物素标记的(dATP)缺口平移PANT在检测短暂大脑中动脉缺血再灌注细胞凋亡与坏死方面的特异性。方法:采用TUNEL,PANT和苏木精-伊红染色方法,检测短暂大脑中动脉缺血30min再灌注不同时间点,前囟水平冠状平面组织切片DNA损伤,采用DNA琼脂糖凝胶电泳,检测细胞核DNA的损伤。结果:大脑中动脉缺血30min以及再灌注2h,DNA琼脂糖凝胶电泳检测到,缺血侧呈细胞坏死特征性条带;苏木精-伊红染色证实纹状体有坏死细胞;但在缺血30min,TUNEL和PANT检测不到阳性细胞。再灌注2h,出现PANT阳性细胞,但邻片TUNEL阳性细胞无增加。再灌注24h,TUNEL,PANT阳性细胞都呈凋亡特征性改变。结论:在短暂局灶性脑缺血再灌注条件下,①再灌注早期,TUNEL和PANT都不标记坏死细胞。②再灌注早期TUNEL不标记DNA单链断裂。③再灌注24h,PANT可标记发生凋亡的单链损伤细胞。④在短暂局灶性脑缺血模型,TUNEL检测细胞凋亡以及PANT检测细胞核DNA单链断裂特异性高。
OBJECTIVE: To investigate the effects of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA polymerase I-mediated biotin-labeled (dATP) nick translocation of PANT in the detection of transient cerebral ischemia-reperfusion Specificity of death and necrosis. Methods: TUNEL, PANT and hematoxylin-eosin staining were used to detect DNA damage in the anterior bland coronal plane tissue sections at different time points after 30 min reperfusion of ischemia of the middle cerebral artery. The DNA was detected by DNA agarose gel electrophoresis DNA damage. Results: Middle cerebral artery occlusion was observed at 30 min after ischemia and 2h after reperfusion. The ischemic area was characterized by necrosis of cells. Hematoxylin and eosin staining showed that there were necrotic cells in the striatum. 30min ischemia, TUNEL and PANT could not detect positive cells. After 2h reperfusion, PANT positive cells appeared, but there was no increase of adjacent TUNEL positive cells. 24h after reperfusion, TUNEL, PANT positive cells showed characteristic changes of apoptosis. Conclusion: Under the condition of transient focal cerebral ischemia-reperfusion, ① Neuro-TUNEL and PANT do not label necrotic cells in the early stage of reperfusion. ② early TUNEL reperfusion without labeling of single-stranded DNA. ③ After reperfusion for 24 h, PANT could be labeled single-stranded damaged cells. ④ In the transient focal cerebral ischemia model, TUNEL detected apoptosis and PANT detection of nuclear DNA single-strand breaks high specificity.