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

缺血和缺氧条件下神经胶质细胞中丝氨酸蛋白酶 mRNA 的估计。

基本信息

批准号：
09671416
负责人：
ANDOH Takashi
金额：
$ 1.86万
依托单位：
ASAHI UNIVERSITY (1998)Gifu University (1997)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671416/
关键词：
Serin protease tissue plasminogen activator r-gsp mRNA ischemia hypoxia PC12 cell apoptosis 虚血プロテアーゼ PLD 分化誘導グリア C6 海馬 mRNA

项目摘要

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.

Caspase是一种与神经细胞死亡密切相关的蛋白酶。另一方面，tPA、uPA和r-gsp这类丝氨酸蛋白酶对神经细胞的作用尚未完全阐明。本研究采用大鼠大脑中动脉（MCA）闭塞模型，以氟烷混合液麻醉，用线栓法阻断大鼠右侧颈内动脉造成局灶性脑缺血，观察缺氧条件下大脑中动脉（MCA）闭塞后脑组织和PC 12细胞中tPA和uPA mRNA表达的变化。将PC 12细胞培养于含10%胎牛血清、5%马血清的培养液中，在37 ℃、含5%CO2的潮湿环境中培养24 h后，置于低氧条件下培养（多气体培养箱;设置为1.0%02，5%C02和余量N2）。分别于缺氧0、3、6、12和24 h后提取总RNA。用RT-PCR技术检测大鼠海马组织中tPA、uPA和r-gsp的mRNA表达，发现缺血6 h后海马组织中tPA和uPA mRNA表达上调。PC 12细胞缺氧6 h后tPA mRNA水平开始升高，并呈节律性变化。缺血状态下r-gsp表达有上调趋势。而uPA mRNA水平无明显变化。提示某些丝氨酸蛋白酶，尤其是tPA和r-gsp可能参与了缺血缺氧后神经元的损伤。