【作者】 张燕；

【导师】 杨晋辉； 尤丽英； 唐映梅； 杨婧；

【作者基本信息】 昆明医学院 ， 内科学， 2010， 硕士

【摘要】 目的:观察氨、谷氨酸培养的SD乳鼠脑星形胶质(astrocytes,AS)细胞水通道蛋白4(aquaporin-4,AQP4)的表达,探讨其和肝性脑病脑水肿发生的关系。以及分别应用地塞米松、二甲基亚砜(Dimethyl sulfoxide,DMSO)和丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPKs)信号转导通路抑制剂U0126观察对其表达的影响,寻求预防和治疗肝性脑病脑水肿的药物。方法:通过氨和谷氨酸培养SD乳鼠脑星形胶质细胞,用倒置相差显微镜和电镜观察细胞形态及超微结构、Fluo-3/Am荧光探针观察细胞内钙离子变化、自动生化分析仪测定LDH漏出量、采用免疫组织化学及逆转录聚合酶链式反应检测AQP4在蛋白和基因水平的表达及其变化,以及分别加用U0126、地塞米松、二甲基亚砜后观察上述指标的变化。结果:氨和谷氨酸可致培养的星形胶质细胞死亡,星形胶质细胞LDH漏出量增加,细胞内钙离子浓度增高,AQP4 mRNA和AQP4蛋白的表达增加。U0126、地塞米松、DMSO可使氨和谷氨酸培养星形胶质细胞LDH漏出明显减少,抑制细胞内钙离子的增加,同时使AQP4 mRNA和AQP4蛋白的表达减少。结论:1、氨和谷氨酸可致培养的星形胶质细胞AQP4表达增加,且随着氨浓度的升高AQP4表达也相应增加。2、U0126、地塞米松、DMSO可以减少星形胶质细胞AQP4的表达,具有细胞保护作用。更多还原

【Abstract】 Objective To investigate the relationship between aquaporin-4(AQP4) and hepatic encephalopathy and to explore the effect of the U0126,a MAPKs signal transduction pathway inhibitor,dexamethasone and dimethyl sulfoxide on the expression of AQP4 in Sprague-Dawley rat astrocytes cultured by the ammonia and glutamate in vitro,providing the theory basis for further research on new medicines to treat hepatic encephalopathy and brain edema.Methods Brain astrocytes in Sprague-Dawley rat were isolated and cultured,and then the morphology and ultra microstructure of cells were observed by inverted phase contrast microscope and transmission electron microscope.Calcium fluorescent indictor of Flluo-3/Am was used to detect the variations of calcium in astrocytes.Automatic chemistry analyzer was used to detect the LDH.The expressional change of AQP4 protein and mRNA was determined by the immunohistochemistry(IHC) and reverse transcription -ploymerase chain reaction.The change of above-mentioned subjects were observed by U0126、dexamethasone and dimethyl sulfoxide.Results Ammonia and glutamate could induce astrocytes death,increasing the level of intracellular free calcium, leading to LDH releasing and increasing the expression of AQP-4 mRNA and protein. U0126、dexamethasone and dimethyl sulfoxide could decrease astrocytes death induced by ammonia and glutamate,reducing cell LDH leakage and decreasing the concentration of calcium.The expression of AQP-4 mRNA and protein could be inhibited with U0126、dexamethasone and dimethyl sulfoxide. Conclusion Ammonia and glutamate can increase the expression of AQP-4,the expression of AQP-4 increasing as long as the increasing of the ammonia.U0126、dexamethasone and dimethyl sulfoxide can decrease the expression of AQP-4,and effectively protect the brain astrocytes.更多还原