胞浆磷脂酶A2参与高血糖加重脑缺血性损伤的初步研究

发布时间：2018-01-13 04:27

本文关键词：胞浆磷脂酶A2参与高血糖加重脑缺血性损伤的初步研究　出处：《宁夏医科大学》2009年硕士论文　论文类型：学位论文

【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。
[Abstract]:Objective to study the neuronal apoptosis and cytoplasmic phospholipase A 2 cytoplasm phospholipase A2 in hippocampal tissue of diabetic rats with hyperglycemia and global cerebral ischemia-reperfusion. To explore the molecular mechanism of hyperglycemia exacerbating cerebral ischemic injury. Methods SD rats were randomly divided into sham-operated control group (Shamma); (2) normal blood glucose cerebral ischemia group (NCI); (3) Diabetic cerebral ischemia group (DCI); 4PD98059 to prevent diabetic cerebral ischemia group (abbreviated as PDG). The model of global cerebral ischemia was established by bilateral common carotid artery ligation and bloodletting. Each ischemic group was subjected to 15 minutes of ischemia and 1 hour of reperfusion. Histopathological Tunel immunohistochemistry and Western blot were used to observe the apoptosis of hippocampal nerve cells. And phosphorylation of MAPK kinase mek) and cPLA2. Results 1) apoptosis of neurons in brain tissue: in NCI group, the majority of neurons in hippocampal CA1, CA2, CA3, CA3, CA4 were apoptotic, except for individual difference. The number of apoptotic cells increased with the prolongation of reperfusion time in most models. Compared with the NCI group, the number of neuronal apoptosis in hippocampal CA1, CA2, CA3, CA3, CA4 and CA1 + CA2 was increased with the prolongation of reperfusion time. The number of neuronal apoptosis in CA3 + CA4 region was increased. In Sham group, hippocampal CA1, CA2 and CA3 were observed. A small number of apoptotic neurons were observed at different time points after global cerebral ischemia-reperfusion in CA4 region. The results of phosphorylated MEK1/2 immunohistochemistry and Western blot showed that diabetic cerebral ischemia was observed for 15 minutes. The expression of phosphorylated MEK1/2 in hippocampal hippocampal CA1, CA2, CA3, CA3 and CA4 at 6 h after reperfusion was observed. Phosphorylated MEK1/2 was expressed at all time points in NCI group, but there was no difference compared with Sham group. The rats in DCI group were subjected to global ischemia for 15 minutes and reperfusion for 1 minute. Apoptosis occurred in almost all neurons in CA2 area of hippocampus at every time point of 6 hours. After PD98059, a specific blocker of MEK1/2, CA1 + CA2 was found in hippocampus. The expression of phosphorylated MEK1/2 was inhibited in CA3 and CA4. At the same time, it can be seen that PD98059 can significantly reduce global cerebral ischemia for 15 minutes and reperfusion for 1 to 3 hours in DCI group at different time points, CA1 and CA2 in hippocampus. Immunohistochemical results of neuronal apoptosis in CA3 and CA4 the hippocampal CA1 + CA2 + CA3 was found at 15 minutes after cerebral ischemia in the group of 10: DCI. The expression of cPLA2 in nerve cells in CA4 region was significantly higher than that in Sham and NCI groups. However, the expression of CPLA2 in hippocampal neurons of CA1, CA2, CA2, CA3, CA4 was significantly higher in the DCI group than in the Sham group at 3 hours after reperfusion, and it was also found. After PD98059 was used, it could reduce global cerebral ischemia for 15 minutes and reperfusion for 1, 3 and 6 hours, at each time point, hippocampal CA1, CA2 and CA2 in the brain tissue of diabetic rats. Expression of cPLA2 in CA3 and CA4 region. Conclusion (1) Diabetic hyperglycemia can aggravate the damage of hippocampal CA1, CA2, CA3 and CA4 during cerebral ischemia-reperfusion. The expression of MEK and cPLA2 increased significantly in diabetic hyperglycemic cerebral ischemia. Diabetic hyperglycemia can increase the activation of ERK1/2 upstream kinase MEK and up-regulate the phosphorylation of ERK1/2 signaling pathway. Brain ischemic damage was aggravated by activating the downstream substrate cPLA2.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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