体外缺血/再灌注神经元DNA损伤的初步研究

STUDY OF DNA DAMAGE WITH NEURONAL ISCHEMIA/REPERFUSION IN VITRO

目的和方法 :应用胎鼠皮层细胞原代培养 ,建立神经元的体外“缺血 /再灌注”模型 ,观察神经元缺血 /再灌注后DNA链的损伤。应用PANT和TUNEL染色分别检测缺血 /再灌注后DNA单链和双链损伤。结果 :神经元缺糖缺氧 2h引起极少量细胞死亡 ,4h引起少于 30 %的细胞死亡 ,而 6～ 8h的缺糖缺氧引起的细胞死亡数量达到80 %以上 ,6h缺糖缺氧再灌注 10～ 18h ,细胞死亡达高峰 ,而在 8h缺糖缺氧再灌注 2h细胞死亡已经达高峰。在缺糖缺氧 2 ,4 ,6 ,8h再灌注 5min ,PANT阳性细胞分别达 30 % ,5 0 % ,80 % ,90 %。而在同样的情况下 ,TUNEL染色阳性细胞数没有明显增加。结论 :体外神经元缺糖缺氧再灌注早期即出现DNA链的损伤 ,且以单链损伤为主。

Aim and Methods: To investigate DNA strand damage in a model of cerebral ischemia/reperfusion injury in vitro . model of ischemia/reperfusion was produced by incubating the primary neuronal cultures to various durations of hypoxia and glucose deprivation(HGD). DNA single and double strand breaks were detectedusing PANT and TUNEL staining respectively. Results: A few cell death occurred after 2 h of HGD. Less than 30 percent of cell died after 4 h of HGD, whereas 6～8 h of HGD resulted in cell death in 80 percent of neurons. Neuronal cell death reached the peak 10～18 h after 6 h of HGD, while it took as early as 2 h after 8 h of HGD. Following HGD, PANT positive cells were remarkably increased, which was propotional to the duration of HGD. At 5 minutes after 2, 4, 6 or 8 h of HGD, PANT positive cells were 30%, 50%, 80%, 90% respectively. Meanwhile, no determined in another cultures, DNA double strain breaks were not significantly increased. Conclution: DNA strain breaks is a very early event of DNA damage following in vitro ischemia/reperfusion, especially DNA single strain breaks.