实验性脑出血大鼠脑内Caspase-3mRNA的表达作用研究

目的动态观察实验性脑出血大鼠脑内血肿周围神经细胞凋亡情况和Caspas-3蛋白及mRNA表达水平的变化，探讨脑H{血后血肿周围神经细胞损伤机制。方法健康雄性Wistar大鼠随机分为生理盐水对照组、假手术组和脑出血模型组，分为术后3h，6h，12h，24h，48h，3d，5d，7d共8个时相点，采用尾状核注射自体非抗凝动脉血复制大鼠脑出血模型，术后制作冰冻切片，对切片进行TUNEL染色，以及Caspase-5免疫组化和原位杂交染色，之后利用图像分析仪，观察阳性细胞数。结果脑出血后3h血肿周围尚无凋亡细胞出现，6h有凋亡发生，以后逐渐增多，3d达高峰后逐渐下降，生理盐水对照组仅有少量TUNEL阳性细胞。假手术组及生理盐水对照组3h无Caspase-3蛋白和mRNA表达，生理盐水对照组6h以后有少量表达，脑出血模型组在6hCaspase-3蛋白和mRNA开始表达，3d时Caspase-3的蛋白达到高峰，5d以后逐渐下降，24hCaspase-3mRNA表达达高峰，5d以后逐渐下降。脑出血后血肿周围脑组织Caspase-3蛋白的表达与TUNEL阳性细胞数呈正相关（r=0．515，P〈0．05）；Caspase-3蛋白表达与mRNA表达呈正相关（r=0．625，P〈0．05）。结论脑出血后血肿周围神经细胞损伤有凋亡机制参与，在出血后6h发生凋亡，第3天达高峰。Caspase-3的表达在Caspase-5蛋白水平变化趋势与脑m血后细胞凋亡的趋势一致，Caspase-3的mRNA水平的表达高峰时间先于Caspase-3蛋白的表达及凋亡的发生，提示Caspase-3的表达决定脑出血后细胞凋亡的发生，在脑出血后细胞凋亡中起促进作用。

The study of the expression of Caspase-3 mRNA in brain after experimental intracerebral hemorrhage in rats

Objective To observe the change of the apotosis and expression level of Caspase-3 protein and mRNA in the brain after experimental intracerebral hemorrhage （ICH） in rats and investigate the mechanism of neuronal injury in intracerebral hemorrhage. Methods The ICH rat model was made by intracranial injection of fresh quantitative autologous blood （by injecting autologous blood slowly in rats ）. Healthy male Wistar rats were randomly divided intonormal saline control groups, sham operation group and ICH model group. Fresh quantitative autologous blood or pure saline solution was injected into caudate nucleus of rat slowly. At 3 h, 6 h, 12 h, 24 h, 3 d, 5 d,7 d after the injection , we got the brain frozen sections. The sections were stained by TUNE, to find the apoptosis cell. The expression of Caspase-3 was observed by immunohistochemistry and in situ hybridization. The number of the positive cells was observed by the image analyzer. Results At 6 hours apoptical cells appeared in the ICH model and were present for more than 14 days, peaking at 3 days. But only a few apoptical cells were found in the normal saline control brains. There were no Caspase-3 protein and mRNA positive cells in the brain of sham operation group and normal saline control group at 3 hours. In ICH model group Caspase-3 protein positive cells and Caspase-3 mRNA positive cells could be found with peaking at 72 hours and 24 hours respectively. Only a few Caspase-3 protein and mRNA positive cells were found in the normal saline control brains. There was positive correlation between the apotosis on the cerebral hemorrhage side of the rats, especially at the perihematoma site, and expression of Caspase-3 protein （r = 0. 513, P 〈 0.05 ）. The expression of Caspase-3 protein correlated positively with the expression of mRNA （ r = 0. 625, P 〈 0.05 ）. Conclusion Apoptosis exists around the hematoma after cerebral hemorrhage. The apoptical neurons appeared at 6 hours peaked at 3 days after hemorrhage. The expression of Caspase-3 protein changed as the apoptosis. The peak time of Caspase-3 mRNA expression preceded that of the expression of Caspase-3 protein. The expression of Caspase-3 decides the level of apoptosis induced by ICH. The pattern of Caspase-3 protein and mRNA play an important role in the process of apotosis of neuron after ICH.