脳虚血後の神経細胞傷害と傷害抑制に関する研究 -アポトーシスおよび非アポトーシス性傷害の検討-

脑缺血后延迟性神经元死亡对细胞凋亡有一定的影响。Ras是参与细胞内信号转导的因子之一，是一种凋亡抑制剂，经pi3激酶和Akt介导。本研究以大鼠前脑缺血再灌注后海马细胞凋亡为基础，探讨Ras在延迟性神经元细胞死亡中的作用。1. #实验方法;采用雄性Wistar大鼠前脑缺血（15分钟）模型。作为缺血状态，双侧颈动脉暂时闭塞后进行无血性低血压。采用HE染色和TUNEL染色进行组织化学研究。免疫印迹法检测大鼠血清中Ras、PI 3-激酶、Akt、Bcl-2及a-fodrin分解产物的含量。缺血-再灌注3 d后，与对照组相比，Ras、PI 3-激酶、Akt、Bcl-2的表达明显降低。a-fodrin的分解产物显著增加。海马神经元细胞HE染色显示脑内细胞核空泡化、固缩，TUNEL染色相同位置呈阳性。2 #实验方法;在前脑缺血（10分钟）模型中，缺血前脑室内注射马霉素A，再灌注后3 d处死。采用HE染色法和TUNEL法对海马进行染色，免疫印迹法检测a-fodrin分解产物的数量。作为对照组，缺血再灌注前脑室内注射生理盐水或2% DMSO。马霉素A注射组海马神经元损伤均小于对照组。结论前脑缺血时Ras和Bcl-2的降解可能在迟发性神经元细胞死亡中调控凋亡，从实验结果来看，海马神经元细胞死亡可能是非凋亡性的。少

It has been noticed that delayed neuronal death after brain ischemia has a certain effect on apoptosis. Ras, one of the factors in intracellular signal transduction, is reported as an inhibitor of apoptosis, intermediating by PI 3-kinase and Akt. In this study, we have investigated the role of Ras in delayed neuronal cell death, based on the presence of apoptosis in hippocampus after ischemia-reperfusion by the model of rat forebrain ischemia.#Experiment 1.METHODS; we utilized the forebrain ischemia (15 minutes) model of male Wistar rat. As ischemic condition, temporary occlusion of bilateral carotid arteries was followed by desanguative hypotension. Histochemical study was performed by HE staining and TUNEL staining. In addition, the amounts of Ras, PI 3-kinase, Akt, Bcl-2, and breakdown product of a-fodrin were surveyed by immunoblotting method.RESULTS; significant decreases of the amounts of Ras, PI 3-kinase, Akt, and Bcl-2 were shown 3 days after ischemia-reperfusion comparing to c … More ontrol group. Breakdown product of a-fodrin increased significantly. Neuronal cells in hippocampus revealed vacuolization and pyknosis of nucleus in the brain sample by HE staining, stained-positive in the same position by TUNEL staining.#Experiment 2METHODS; in the forebrain ischemia (10 minutes) model, manumycin A was injected intracerebroventricularly before ischemia, and sacrificed 3 days after reperfusion. The hippocampi were stained by HE staining and TUNEL method, the quantities their breakdown products of a-fodrin were measured by immunoblotting. As the control group, physiological saline or 2% DMSO were injected intracerebroventricularly before ischemia-repersion.RESULTS; manumycin A injected group revealed that the hippocampal neurons injured less than the control group in every methods.#CONCLUSIONSThe degradation of both Ras and Bcl-2 in forebrain ischemia might suggest the regulation of apoptosis in delayed neuronal cell death, the neuronal cell death in hippocampus might be non-apoptotic judging from the result of the experiment 2. Less