Estimation of serine protease mRNA in glial cells after ischemic and hypoxic conditions.

众所周知，Caspase是一种Cistein蛋白水解酶，与神经细胞死亡有关。另一方面，tPA、uPA和r-GSP这三种丝氨酸蛋白酶在神经细胞中的作用尚未完全阐明。本实验观察了低氧状态下大鼠大脑中动脉(MCA)闭塞后tPA和uPA mRNA表达的变化以及PC12细胞在低氧条件下的变化。于大脑中动脉结扎6h处死大鼠，提取双侧大脑半球海马区总RNA。PC12细胞系在37ﾟC含5%二氧化碳的湿空气中加入10%胎牛血清、5%马血清维持培养，在含2%胎牛血清的培养液中孵育24小时后，置于低氧条件下(多气体孵箱；设置为1.0%02，添加5%二氧化碳，其余为氮气)。分别于缺氧后0、3、6、12、24小时提取总RNA。分别用tPA、uPA和r-GSP的特异性引物进行RT-PCR分析，观察大鼠脑缺血6h后各时间点tPA和uPA mRNA表达的变化。PC12细胞在缺氧6h后，tPA基因表达水平逐渐升高，并出现明显的细胞凋亡改变。在缺血状态下，r-GSP表达有上调趋势。而uPA基因表达水平无明显变化。这些结果提示，一些丝氨酸蛋白酶，特别是tPA或r-GSP可能在缺血或缺氧后的神经元损伤中起作用。

It is well-known that Caspase, kinds of Cistein proteases, are concerned in neuronal cell death. On the other hand, the roles of tPA, uPA and r-gsp, kinds of Serin proteases, for neuronal cells have not yet been fully clarified. We investigated changes in tPA and uPA mRNA expressions in the rat brain after middle cerebral artery (MCA) occlusion and PC12 cells under hypoxic condition.Focal cerebral ischemia was produced by MCA occlusion using an intraluminal suture inserted from right internal carotid artery in male Sprague-Dawley rats (280 to320 g) under anesthesia with a mixture of fluothane. After rats were decapitated at 6 hours of MCA occlusion, total RNA were extracted from each hippocampus of bilateral hemispheres.PC12 cell line was maintained in medium supplemented with 10% FBS, 5% horse serum in a humidified atmosphere containing 5% C02 at 37ﾟC.After 24h of incubation in the medium containing 2% FBS, cells were placed into hypoxic condition (multigas incubator ; set to 1.0% 02, with 5% C02 and the balance N2). Total RNA were extracted after exposure of hypoxia for 0, 3, 6, 12 and 24 hours. Extracted RNAs were analysed by RT-PCR technique using specific primer of tPA, uPA and r-gsp for investigation of changes in these mRNA expressions in each period.Up-regulation of tPA and uPA mRNA were revealed in the rat hippocampus following 6 hours ischemia. The level of tPA mRNA was increased from the period of 6 hours exposure of hypoxia in PC12 cell, rustled in apoptoic changes. There is a tendency of up-regulation of r-gsp expression in ischemic condition. However, the level of uPA mRNA was not revealed significant changes. These results suggest that some of Serine proteases, especially tPA or r-gsp may play a role in the neuronal damage after ischemia or hypoxia.