放线菌酮连续灌注对大鼠脑缺血后DNA片段形成的影响

Effect of Cycloheximide Continuous Injection on DNA Fragmentation after Focal Cerebral Ischemia in Rats

目的揭示蛋白合成抑制剂对脑缺血的不同时期脑细胞凋亡的保护作用。方法将wistar大鼠制成MCA缺血—再灌注模型,分成放线菌酮(CHX)组、假手术组和生理盐水(对照)组。放线菌酮组从缺血前15min开始连续测脑室放线菌酮灌注(1mg/kg·d-1),于缺血60min后再灌注l、3、7、14d处死动物,取脑冠状切面,用TUNEL染色,DNA电泳观察细胞凋亡现象。对照组则用复方氯化钠溶液连续灌注。结果CHX组在l、3d时间分别发现(158±16)、(86±10)个阳性细胞。Tunel阳性细胞与对照组无明显差异。第7、14dCHX组TUNEL阳性细胞显著低于对照组(P<0.05),CHX组在缺血l、3、7d发现DNA梯带,第14d未发现DNA梯带形成;对照组4次均出现DNA梯带。结论脑梗塞过程一直伴随DNA片段形成。在早期,蛋白生成抑制剂似乎不能阻止DNA片段的形成,而在晚期则能有效地阻止TUNEL阳性细胞的出现。缺血早期DNA片段形成可能不依赖蛋白质合成,而1周以后,蛋白质合成才成为DNA片段形成的前提。

Objective To evaluate the kinetic and protective effect of protein synthesis inhibitor cycloheximide on the brain cells apoptosis in various stages of cerebral ischemia.Methods 36 Wister rats' middle cerebral artery (MCA) were occluded for 60 minutes, reperfused for 24 hours, 72 hours, 7 days, 14 days. Cycloherximide was injected continuous through ventriculus lateralis since 15 minutes before cerebral ischemia in therapy group (1mg. kg -1 . d -1 ). DNA fragmentation evaluated by mines of TUNEL method and DNA electrophoresis. Results The results is showed that: after 24,72 hours of reperfusion, there were no significant difference of TUNEL positive nuclei between CHX and control groups. After 7, 14 days, the DNA fragmentation in the inner boundary zone of CHX group were less than that of control group.Conclusion It's indicated that DNA fragmentation accompanies ischemic cell death. In acute stage, the fragmentation didn't rely on the synthesis of protein, and after acute stage protein synthesis is the essential factor of DNA fragmentation. It may be another kind of cell death which like apoptosis in some extent. It could be named as Apoptosis like Cell Death.