磷酸化细胞外信号转导通路介导高血糖加重脑缺血再灌注损伤星形胶质细胞凋亡的初步研究

发布时间：2018-05-23 12:19

本文选题：高血糖 + 脑缺血　；参考：《宁夏医科大学》2010年硕士论文

【摘要】：目的研究急性高血糖状态下,观察大鼠全脑缺血再灌注模型大脑扣带皮质区和海马CA4区星形胶质细胞的凋亡,以及p-ERK及其上游激酶MEK、下游作用底物cPLA2的激活表达,探讨星形胶质细胞在高血糖加重全脑缺血再灌注损伤中的作用和可能分子机制。方法SD大鼠随机分为①假手术对照组(简称Sham);②正常血糖脑缺血组(简称NCI);③糖尿病脑缺血组(简称DCI)。采用双侧颈总动脉结扎并放血法制备全脑缺血模型,各缺血组再按照缺血15min、再灌注1h、3h、6h亚组观察。采用组织病理学,TUNEL、免疫组织化学双重标记,Western blot等方法,对比观察大脑扣带皮质区和海马CA4区星形胶质细胞的凋亡,p-ERK及其上游激酶p-MEK、下游作用底物cPLA2的磷酸化状态。结果(1)脑组织星形胶质细胞凋亡:NCI组在缺血15min、再灌注1、3、6h各时间点扣带皮质区和海马CA4区内星形胶质细胞凋亡数增多,并随缺血再灌注时间的逐渐延长而增多,明显高于Sham组;DCI组在全脑缺血15min、再灌注1、3、6h各时间点大脑扣带皮质区和海马CA4区内凋亡星形胶质细胞数明显增多,高于Sham组和NCI组。(2)磷酸化ERK1/2免疫组化表达情况:NCI组扣带皮质区和海马CA4区内在全脑缺血15min、再灌注1、3、6h各时间点仅见个别p-ERK阳性表达的星形胶质细胞,与Sham组相比无差异;DCI组扣带皮质区和海马CA4区内p-ERK阳性表达的星形胶质细胞数明显增多,高于Sham和NCI组。(3)磷酸化MEK免疫组化表达情况:NCI组扣带皮质区和海马CA4区内在各观察时间点仅见少量p-MEK阳性表达的星形胶质细胞,扣带皮质和海马CA4区内均于再灌注6h达高峰,高于Sham组;DCI组大脑扣带皮质区和海马CA4区内p-MEK阳性表达星形胶质细胞数增多,扣带皮质区内于再灌注6h达高峰,海马CA4区内于再灌注3h达高峰,高于Sham组和NCI组。(4)cPLA2免疫组化结果:NCI组扣带皮质区和海马CA4区内在各观察时间点仅见个别散在分布的cPLA2阳性表达的星形胶质细胞,与Sham组相比无差异;与Sham组和NCI组相比,DCI组扣带皮质和海马CA4区内阳性表达细胞数稍有增加。结论(1)高血糖能够加重脑缺血再灌注时扣带皮质和海马CA4区损伤;(2)神经细胞和星形胶质细胞凋亡增加参与了高血糖加重脑扣带皮质和海马CA4区缺血再灌注损伤;(3)高血糖脑缺血时,星形胶质细胞凋亡能通过促进神经元凋亡加重脑缺血性损伤;(4)高血糖脑缺血时ERK1/2介导了扣带皮质区和海马CA4区神经细胞和星形胶质细胞凋亡的增加;(5)上游激酶MEK活化高表达、激活ERK1/2,从而引起下游作用底物cPLA2表达增加,参与了扣带皮质区和海马CA4区神星形胶质细胞凋亡增加。
[Abstract]:Objective to investigate the apoptosis of astrocytes in cingulate cortex and hippocampal CA4 in rats with acute hyperglycemia, and to investigate the activation and expression of p-ERK and its upstream kinase MEK, the downstream substrate cPLA2. To investigate the role and possible molecular mechanism of astrocytes in the exacerbation of global cerebral ischemia reperfusion injury with hyperglycemia. Methods Sprague-Dawley rats were randomly divided into 1 sham-operated control group (Shambun 2) normal blood glucose cerebral ischemia group (NCI) 3 diabetic cerebral ischemia group (DCI). The model of global cerebral ischemia was established by bilateral common carotid artery ligation and bloodletting. Each ischemic group was observed according to ischemia for 15 min and reperfusion for 1 h for 3 h and 6 h for subgroup. The apoptosis of astrocytes in the cingulate cortical area and the hippocampal CA4 area was observed by using histopathological Tunel and immunohistochemical double labeling Western blot. The phosphorylation of the downstream substrate cPLA2 and the upstream kinase p-MEK were observed. Results 1) the apoptosis of astrocytes in brain tissue increased at 15 min after ischemia and 1 h after reperfusion for 6 h, and the number of apoptotic astrocytes in the cingulate cortex and hippocampal CA4 increased with the prolongation of ischemia-reperfusion time. The number of apoptotic astrocytes in cerebral cingulate cortex and hippocampal CA4 was significantly higher in Sham group than that in Sham group at 15 min after global cerebral ischemia and 1 h and 3 h after reperfusion at 6 h after reperfusion, and the number of apoptotic astrocytes in the cingulate cortex and hippocampal CA4 was significantly increased. The expression of phosphorylated ERK1/2 was higher than that in Sham group and NCI group. The expression of phosphorylated ERK1/2 in the cingulate cortical area and hippocampal CA4 area was observed at 15 min after global cerebral ischemia, and only a few astrocytes with p-ERK positive expression were observed at 1h and 3h after reperfusion at 6 h after reperfusion. The number of astrocytes with p-ERK positive expression in cingulate cortical area and hippocampal CA4 area in Sham group was significantly higher than that in Sham group. The expression of phosphorylated MEK was higher than that in Sham and NCI groups. The expression of p-MEK in the cingulate cortex and hippocampal CA4 was only a small amount of astrocytes in the CA4 area of hippocampus and cingulate cortex, and reached the peak at 6 h after reperfusion in both the cingulate cortex and the hippocampal CA4 area. Compared with Sham group, the number of p-MEK positive cells in cingulate cortical area and hippocampal CA4 area increased, cingulate cortex reached its peak at 6 h after reperfusion, and hippocampal CA4 region reached its peak at 3 h after reperfusion. The immunohistochemical results were higher than those in Sham group and NCI group. The results of immunohistochemistry showed that there were only a few scattered cPLA2 positive astrocytes in the cingulate cortex and hippocampal CA4 area in the Sham group and the NCI group, but there was no difference compared with that in the Sham group. Compared with Sham group and NCI group, the number of CA4 positive cells in cingulate cortex and hippocampus increased slightly. Conclusion (1) hyperglycemia can aggravate the damage of cingulate cortex and hippocampal CA4 area during cerebral ischemia-reperfusion.) the increase of apoptosis of neurons and astrocytes is involved in hyperglycemia exacerbating cerebral cingulate cortex and hippocampal CA4 ischemia-reperfusion injury. During hyperglycemic cerebral ischemia, Apoptosis of astrocytes can increase apoptosis of neurons and astrocytes in the cingulate cortex and hippocampal CA4 region by promoting neuronal apoptosis and exacerbating ischemic brain injury. The expression of MEK activation in upstream kinase is higher than that in hyperglycemic cerebral ischemia, and ERK1/2 mediates the apoptosis of neurons and astrocytes in the cingulate cortical and hippocampal CA4 regions. Activation of ERK1 / 2 resulted in increased expression of substrates cPLA2 and increased apoptosis of astrocytes in the cingulate cortex and hippocampal CA4.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

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