脑缺血后PUNT的表达及其与DNA合成标记物的关系

Expression of PUNT Positive Cells and Its Association with DNA Synthetical Marker after Cerebral Ischemia

目的 研究脑缺血后DNA的损伤及其与DNA合成的标记物——溴代尿嘧啶脱氧核苷(bromodeo—xyuridine,BrdU)掺入的关系。方法 采用 PUNT(in situ DNA polymerase I-mediated DIG-dUTP nick-transla-tion)染色方法检测DNA的单链断裂(single strand break,SSB),用免疫荧光双标法观察BrdU的掺入与DNA损伤的关系。结果(1)PUNT染色阳性细胞表达的时程变化显示,缺血缺氧(hypoxic-ischemia,H-I)后0.5h,缺血侧纹状体就可检测到PUNT染色阳性细胞的表达。除H-I后3 h阳性细胞数出现一个小高峰外,H-I后1到12h,阳性细胞数处于较低水平。从H-I后24 h起,阳性细胞数显著上升,到2d时达最高峰。H-I后4和6 d,阳性细胞数仍然维持在较高的水平。(2)H-I后2到6d,BrdU与PUNT染色阳性细胞共表达。结论 H-I后早期出现DNA的损伤,BrdU的掺入与DNA的损伤有关。

Purpose: To explore DNA injury and its association with the marker of DNA synthesis, bromodeoxyuridine (BrdU) incorporation after cerebral ischemia. Methods: PUNT (in situ DNA polymerase I-mediated DIG-dUTP nick translation) staining was employed to detect DNA single strand breaks(SSB) and immunofluorescent double staining to clarify the relationship of BrdU incorporation with DNA injury. Results: (1)The time course of PUNT expressed cells showed positive cells occurred in the ischemic striatum as early as 0.5 h after hypoxic-ischemia (H-I). From 1 to 12 h, the number of positive cells persisted in a relatively lower lever except a small peak at 3 h after H-I. From 24 h on, the number of positive cells increased markedly, attained a peak value at 2 d, and still remained a higher level at 4 and 6 d after H-I. (2) Results from immunofluorescent double staining revealed that BrdU was co-stained with PUNT in the ischemic striatum from 2 to 6 d after H-I. Conclusions: DNA damage occurs earlier after H-I and BrdU incorporation is related to DNA damage.