【作者】 王伟伟；

【导师】 康劲松；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 目的:通过观察糖尿病时脑皮质水通道AQP4 mRNA、氯离子通道蛋白CLC-2和CLIC4、凋亡相关蛋白Bax和Bcl-2水平表达变化,探讨糖尿病时脑损伤的机制。方法:将大鼠随机分为非糖尿病组和糖尿病组。50mg/kg链脲佐菌素(streptozotocin,STZ)行左下腹腔一次性注射复制糖尿病模型。注射STZ后第72h,监测血糖,血糖大于16.7mmol/L,且有多饮、多食、多尿现象者,确定为糖尿病大鼠模型复制成功。模型建立成功后,常规饲养,每周称量体重,隔周检测血糖,共监测8周。第九周再次检测血糖,达到16.7mmol/L或以上者进入分组,未达到血糖指标者不进入分组。HE染色法观察大脑皮质的形态学改变;RT-PCR检测大脑皮质中水通道AQP4 mRNA的表达;Western blotting检测大脑皮质中Bax、Bcl-2、CLC-2和CLIC4的蛋白表达。结果:与非糖尿病组大鼠相比,糖尿病组大鼠血糖升高;神经元出现核质浓缩着色较深,胞体固缩等形态学改变;脑皮质水通道AQP4 mRNA水平下降;Bax/Bcl-2的比值明显升高;CLC-2、CLIC4的蛋白表达明显增强。结论:糖尿病时脑皮质出现损伤,水通道AQP4 mRNA表达下调,使脑内水分的清除受阻,加重糖尿病脑损伤。糖尿病时脑皮质CLC-2、CLIC4蛋白的表达增高通过线粒体途径引起大脑皮质神经细胞凋亡,造成脑损伤。更多还原

【Abstract】 Objective:To investigate the mechanism of brain injury induced by diabetes through observing the changes of AQP4 mRNA and protein CLC-2、CLIC4、Bax、Bcl-2 in the cerebral cortex.Method:1 Wistar rats were divided into two groups randomly:the non-diabetic rats (NDM group,control group) and the diabetic rats(DM group). The diabetic rats were dealt with left lower abdominal cavity injection of streptozotocin(STZ) 50 mg per kg.After three days, blood glucose in tail-vein blood was measured by blood glucose monitor,those whose blood glucose were 16.7mmol/l or much higher as well as polydipsia、polyphagia、diuresis were testify as diabetic rats.After the model established,reared routinely,body weight was measured weekly and blood glucose fortnightly,altogether 8 weeks.Then blood glucose was measured again, whose blood glucose were 16.7mmol/l or much higher were classified as diabetes. Put the rats to death and decapitated,then segregated the cerebral cortex,put them in the liquid nitrogen for quick freezing and then preserved them in the circumstance of subzero 80 degree.2 The measurement of body weight and the measurement of blood glucose during the experiment processes.3 The examation of cerebral cortex through hematoxylin-eosin staining.4 RT-PCR was used to detect AQP4 mRNA expression in cerebral cortex.5 Western blotting was used to analyze the expression of Bax、Bcl-2、CLC-2 and CLIC4 in cerebral cortex.Results:1 The changes of body weight and blood glucose between anterior and posterior of the experiment.After intraperitoneal injection of STZ,compared with the NDM group the body weights of the DM group rats decreased abviously;The blood glucoses of the NDM and DM group rats were uniformity before the experiment,and the blood glucose of the DM group rats maintained hyperglycosemia status after intraperitoneal injection of STZ,while they didn’t change in the NDM group rats.2 Hematoxylin-eosin staining of cerebral cortex from diabetic rats.The distribution of eurons and neuroglial cells in NDM group was no abnormality,the body of neuron with little apophysis was ellipse,the nucleus was round and the nucleoli distinct;The tissue organization of cerebral cortex in DM group was rarefaction, nucleoplasm condense, cell body pyknosis,and the form of neuron changed to polygon or fan-shape.The result of hematoxylin-eosin staining suggest the damage of brain induced by diabetes.3 The expression of AQP4 mRNA in the cerebral cortex of the DM group rats.Compared with the NDM group,the expression of AQP4 mRNA in DM group decreased abviously. The changes of AQP4 is correlated with brain edema closely,the downregulation of AQP4 mRNA might involved in the generation of Vasogenic edema,which resulted in brain injury.4 The expression of Bax and Bcl-2 protein in the cerebral cortex of the DM group rats.Compared with the NDM group,the expression of Bax protein increased and the expression of Bcl-2 protein decreased in the DM group,the ratio of Bax/Bcl-2 increased abviously.The family of Bcl-2 protein is the crucial regulation protein during the apoptosis of mitochondrial pathway,Bax is the pro-apoptosis protein,while Bcl-2 is the anti-apoptosis protein.The rise of the ratio of Bax and Bcl-2 indicated that the apoptosis of mitochondrial pathway involved in the brain injury induced by diabetes.5 The expression of CLC-2 protein in the cerebral cortex of the DM group rats.Compared with the NDM group,the expression of CLC-2 protein increased abviously in the DM group. AS the voltage gating chloride channel,the upregulation of CLC-2 might cause the increase of CL- outflow,result in apoptosis and involved in brain injury induced by diabetes.6 The expression of CLIC4 protein in the cerebral cortex of the DM group rats.Compared with the NDM group,the expression of CLIC4 protein increased in the DM group.The upregulation of CLIC4 might involve in the loss of mitochondrial membrane potentials,the release of Cyt-C,the activation of caspase and the upregulation of Bax,result in apoptosis through the mitochondrial pathway,which involved in the brain injury induced by diabetes.Conclusion:1 The blood glucose increased abviously and the body weight decreased abviously compared with NDM group indicated the model of diabetic rats were established successfully.2 The hematoxylin-eosin staining of cerebral cortex revealed the degenerative changes of nerve cells,which is thus evident that the brain damage induced by diabetes.3 In the cerebral cortex of the DM group rats,the expression of AQP4 mRNA decreased compared with NDM group,which might hinder the clearance of water in the brain and involve in the generation of Vasogenic edema,thus aggravated brain injury induced by diabetes.4 The abvious increase of CLC-2 and CLIC4 in the cerebral cortex of the DM group rats consistently with the increase of the ratio of Bax/Bcl-2 revealed that diabetes might induce apoptosis of nerve cells through the mitochondria,which may thus result in brain injury induced by diabetes.更多还原