【作者】 常越；

【导师】 张建中； 景丽；

【作者基本信息】 宁夏医科大学 ， 病理学与病理生理学， 2009， 硕士

【摘要】 目的研究糖尿病高血糖全脑缺血再灌注大鼠模型海马组织神经细胞凋亡及胞浆磷脂酶A2(cytoplasm phospholipase A2, cPLA2)的激活表达,探讨高血糖加重脑缺血性损伤的分子机制。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);④PD98059预防糖尿病脑缺血组(简称PD)。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、免疫组织化学和蛋白印迹等方法,对比观察海马神经细胞的调亡、以及MAPK激酶(MEK)和cPLA2的磷酸化状态。结果(1)脑组织神经元凋亡: NCI组海马CA1、CA2、CA3、CA4区多数神经细胞发生凋亡,除个体差异影响外,大多数模型随着再灌注时间的逐渐延长凋亡细胞数增加;与NCI组相比,DCI组海马CA1、CA2、CA3、CA4区神经细胞发生凋亡的数目增多,并随着再灌注时间的逐渐延长海马CA1、CA2、CA3、CA4区神经细胞发生调亡的数目增加;而Sham组脑组织海马CA1、CA2、CA3、CA4区全脑缺血再灌注各时间点可见少量凋亡神经细胞。(2)磷酸化MEK1/2免疫组化及Western blot结果:观察糖尿病脑缺血全脑缺血15分钟,再灌注1、3、6小时各时间点脑组织海马CA1、CA2、CA3、CA4区神经细胞磷酸化MEK1/2的表达情况,结果发现,在NCI组磷酸化MEK1/2在各时间点均有表达,但与Sham组相比无差异;DCI组在全脑缺血15分钟、再灌注1、3、6小时各时间点脑组织海马CA2区几乎所有神经细胞都发生了凋亡改变。采用MEK1/2的特异性阻断剂PD98059后,海马CA1、CA2、CA3和CA4各区神经细胞磷酸化MEK1/2表达受到抑制;同时也可见使用PD98059后,能够明显减少DCI组全脑缺血15分钟、再灌注1、3、6小时各时间点脑组织海马CA1、CA2、CA3和CA4各区神经细胞凋亡。(3)cPLA2免疫组化结果:DCI组全脑缺血15分钟时,海马CA1、CA2、CA3、CA4区中神经细胞cPLA2的表达情况与Sham组和NCI组比较阳性表达显著增加;而在再灌注3小时,cPLA2在海马CA1、CA2、CA3、CA4区神经细胞的表达结果显示,DCI组比Sham组明显增多。同时也发现,在使用PD98059后,能够减少糖尿病脑缺血全脑缺血15分钟、再灌注1、3、6小时各时间点脑组织海马CA1、CA2、CA3和CA4区cPLA2的表达。结论(1)糖尿病高血糖能够加重脑缺血再灌注时海马CA1区、CA2区、CA3区、CA4区损伤,导致凋亡神经细胞明显增加。(2)糖尿病高血糖脑缺血时MEK和cPLA2激活表达显著增加,可能与海马各区神经细胞凋亡增加有关。糖尿病高血糖能够导致ERK1/2上游激酶MEK激活增加,使ERK1/2信号通路磷酸化上调,通过激活下游作用底物cPLA2加重了脑缺血性损伤。更多还原

【Abstract】 Objective We studied the apoptosis of nerve cells and the expression of activation of cytoplasm phospholipase A2 (cPLA2) in the hippocampus in the global cerebral ischemia-reperfusion rat models, and explored the molecular mechanism of hyperglycosemia- aggravated ischemic brain injury.Methods Spraggue Dawle rats were randomized into four groups:①Sham operation groups(Sham);②Normoglycemic cerebral ichemia groups (NCI);③Diabetes cerebral ichemia groups (DCI);④PD98059 prevented diabetes cerebral ichemia groups (PD)。The making method of global cerebal ischemia model: ligated bilateral carotid artery and withdraw blood. Each ischemic group divided into four sub-groups, ischemia 15min, reperfusion 1h, 3h, 6h. Histopathology, TUNEL, Immunohistochemistry, Weatern blotting techniques methods were used to observe the information of neural cell apoptosis and the phosphorylation of MEK1/2 and cPLA2 in hippocampus CA1, CA2, CA3 and CA4 region.Result (1) Neuronal apoptosis of brain: In the NCI groups the majority of nerve cells happen apoptosis, and with the reperfusion time extended most models gradually increase in the number of apoptotic cells in the hippocampus CA1, CA2, CA3 and CA4 region. Compare with the NCI groups, the number of apoptotic nerve cells in the DCI groups markedly increased in the hippocampus CA1, CA2, CA3 and CA4 region, and with the reperfusion time gradually extended the number of apoptotic nerve cells gradually increase. In the Sham groups, we can see a little of apoptotic nerve cells at different time points of reperfusion in the hippocampus. (2) Phosphorylation of MEK1/2 by immunohistochemistry and Western blot results:In the diabetic cerebral ischemia global cerebral ischemia medols at 15 minutes after ischemia and 1,3,6-hour after reperfusion in hippocampal CA1, CA2, CA3, CA4 region the result of expression phospho-MEK1/2 of nerve cells: In the NCI groups we found phospho-MEK1/2 had expressed at different time points, but there was no difference compared with the Sham groups. In the diabetic cerebral ischemia global cerebral ischemia medols at 15 minutes after ischemia and 1,3,6-hour after reperfusion in hippocampal CA2 region, nerve cell mostly happen apoptosis. The use of MEK1/2 specific inhibitor PD98059, in the hippocampal CA1, CA2, CA3 and CA4 phospho-MEK1/2 expression of nerve cell was inhibited. In the diabetic cerebral ischemia global cerebral ischemia medols at 15 minutes after ischemia and 1,3,6-hour after reperfusion in hippocampal CA1, CA2, CA3, CA4 region, PD98059 can reduce the number of apoptotic nerve cells. (3) Immunohistochemical results of cPLA2: In the diabetic cerebral ischemia global cerebral ischemia medols at 15 minutes after ischemia and 1,3,6-hour after reperfusion in hippocampal CA1, CA2, CA3, CA4 region the result of expression cPLA2 of nerve cells: Compared with Sham groups and NCI groups the expresson of cPLA2 significantly increase. At 3h after reperfusion in hippocampal CA1, CA2, CA3, CA4 region the expresson of cPLA2 significantly increased compared with Sham groups. We also found that after the use of PD98059, the expression of cPLA2 was inhibited at 15 minutes after ischemia and 1,3,6-hour after reperfusion in hippocampal CA1, CA2, CA3, CA4 region.Conclusions (1) Diabetes-induced hyperglycaemia can aggravate the injury of CA1, CA2, CA3, CA4 region of hippocamp and leaded to apoptotic nerve cells significantly increase, as cerebral ischemia-reperfusion. (2) As diabetes-induced hyperglycaemia cerebral ischemia, the activation of MEK and cPLA2 incrase. This may be related to the incrase of nerve cells apoptosis in hippocamp. Diabetes-induced hyperglycaemia can induced the expression of MAPK kinase (MEK) that is ERK1/2 upstream kinase significantly increase and leaded to the up-regulation of phosphorylation-ERK1/2-signaling-pathway. The activation of cPLA2 that is ERK1/2’s downstream substrate aggravate ischemic brain injury.更多还原