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目的构建新生儿缺氧缺血性脑病的经典动物模型,并分析在缺氧缺血条件下,促红细胞生成素(EPO)治疗前后胶质细胞谷氨酸运载蛋白2(EAAT2)表达情况。方法 108只出生72h的SD乳鼠随机分为六组:空白对照组、单纯缺氧组、单纯缺血组、缺氧缺血组、促红细胞生成素治疗组和生理盐水治疗组。缺氧缺血性脑病模型通过双侧结扎颈动脉并且缺氧处理2h建立。观察并记录不同实验组中乳鼠行为的变化。利用蛋白质印迹技术从蛋白质水平上分别检测不同实验组中EAAT2蛋白的表达情况。利用实时荧光定量聚合酶链反应(PCR)技术从mRNA水平上分别检测不同实验组中新生乳鼠的海马区、大脑皮层EAAT1、EAAT2的基因表达情况。结果模型呈现典型的缺氧缺血脑病特征。缺氧、缺血、缺氧缺血都可以导致海马组织、大脑皮层中EAAT1的mRNA水平及EAAT2表达量的降低。经过EPO治疗后,EAAT1及EAAT2的基因表达量明显上升。并且,大脑皮层中基因表达水平要高于海马组织中基因表达水平。结论 EAAT2表达量的下降可能是HIE发病的一个原因,EPO的疗效可能是通过上调EAAT2的表达量来实现。
Objective To construct a classical animal model of neonatal hypoxic-ischemic encephalopathy and analyze the expression of glutamate transporter 2 (EAAT2) in glial cells before and after erythropoietin (EPO) treatment under hypoxic-ischemic conditions. Methods A total of 108 SD infants born 72h were randomly divided into six groups: control group, hypoxia group, ischemia group, hypoxia-ischemia group, erythropoietin-treated group and saline-treated group. Hypoxic ischemic encephalopathy model was established by bilateral carotid artery ligation and hypoxia 2h. Observed and recorded changes in different experimental groups suckling mice behavior. Western blotting was used to detect the expression of EAAT2 protein in different experimental groups from the protein level. The mRNA expression of EAAT1 and EAAT2 in hippocampus and cerebral cortex of neonatal neonatal rats in different experimental groups were detected by real-time fluorescence quantitative polymerase chain reaction (PCR). Results The model showed typical characteristics of hypoxic-ischemic encephalopathy. Hypoxia, ischemia and hypoxia-ischemia all can lead to the decrease of EAAT1 mRNA and EAAT2 expression in hippocampus and cerebral cortex. After EPO treatment, EAAT1 and EAAT2 gene expression increased significantly. Moreover, the level of gene expression in the cerebral cortex is higher than that in the hippocampus. Conclusions The decrease of EAAT2 expression may be one of the causes of HIE. The efficacy of EPO may be achieved by up-regulating EAAT2 expression.