短暂局灶性脑缺血再灌注条件下细胞凋亡与坏死动态变化的特异性评价

Evaluation on specificity of dynamic changes of apoptosis and necrosis of cells following transient focal cerebral ischemia-reperfusion

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。

AIM:To investigate the specificity of terminal deoxynucleotide transferase- mediated dUTP nick- end labeling(TUNEL) and polymerase I- mediated biotin- datp nick translation(PANT) related to the determination of apoptosis and necrosis of cells in transient focal middle cerebral artery ischemia- reperfusion in order to provide academic reference for the recovery of neural function after cerebral ischemia.METHODS:TUNEL,PANT and hematoxylin and eosin(HE) staining were used to detect the coronal sections located at frontal fontanelle at different times of reperfusion after 30- minute middle cerebral artery ischemia- reperfusion,and the damage of DNA in nucleus was detected with DNA agarose gel electrophoresis. RESULTS:After 30- minute middle cerebral artery ischemia and 2- hour reperfusion,the results of DNA agarose gel electrophoresis showed indicative occurrence of cell necrosis which also found with HE staining, but TUNEL and PANT exhibited no staining.After 2- hour reperfusion,PANT positive cells appeared while TUNEL positive cells did not increase.Either TUNEL or PANT stained nuclear exhibited typical modality of apoptosis after 24- hour reperfusion. CONCLUSION:On the condition of transient focal cerebral ischemia:① Neither TUNEL nor PANT labeled necrotic cells exist at early reperfusion;② There are no TUNEL- labeled DNA single- strand breaks;③ PANT labeled cells with apoptotic modality are found after 24 hours of reperfusion;④ TUNEL detection exhibits high specificity to apoptosis cells, and PANT detection exhibits high specificity to single- strand breaks.