本文關(guān)鍵詞：胞漿磷脂酶A2參與高血糖加重腦缺血性損傷的初步研究　出處：《寧夏醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 高血糖 腦缺血 凋亡 胞漿磷脂酶A2 細(xì)胞外信號調(diào)節(jié)激酶 絲裂原活化蛋白激酶激酶

【摘要】： 目的研究糖尿病高血糖全腦缺血再灌注大鼠模型海馬組織神經(jīng)細(xì)胞凋亡及胞漿磷脂酶A2(cytoplasm phospholipase A2, cPLA2)的激活表達(dá),探討高血糖加重腦缺血性損傷的分子機(jī)制。 方法SD大鼠隨機(jī)分為①假手術(shù)對照組(簡稱Sham);②正常血糖腦缺血組(簡稱NCI);③糖尿病腦缺血組(簡稱DCI);④PD98059預(yù)防糖尿病腦缺血組(簡稱PD)。采用雙側(cè)頸總動脈結(jié)扎并放血法制備全腦缺血模型,各缺血組再按照缺血15min,再灌注1h、3h、6h亞組觀察。采用組織病理學(xué)、TUNEL、免疫組織化學(xué)和蛋白印跡等方法,對比觀察海馬神經(jīng)細(xì)胞的調(diào)亡、以及MAPK激酶(MEK)和cPLA2的磷酸化狀態(tài)。 結(jié)果(1)腦組織神經(jīng)元凋亡: NCI組海馬CA1、CA2、CA3、CA4區(qū)多數(shù)神經(jīng)細(xì)胞發(fā)生凋亡,除個體差異影響外,大多數(shù)模型隨著再灌注時間的逐漸延長凋亡細(xì)胞數(shù)增加;與NCI組相比,DCI組海馬CA1、CA2、CA3、CA4區(qū)神經(jīng)細(xì)胞發(fā)生凋亡的數(shù)目增多,并隨著再灌注時間的逐漸延長海馬CA1、CA2、CA3、CA4區(qū)神經(jīng)細(xì)胞發(fā)生調(diào)亡的數(shù)目增加;而Sham組腦組織海馬CA1、CA2、CA3、CA4區(qū)全腦缺血再灌注各時間點可見少量凋亡神經(jīng)細(xì)胞。(2)磷酸化MEK1/2免疫組化及Western blot結(jié)果:觀察糖尿病腦缺血全腦缺血15分鐘,再灌注1、3、6小時各時間點腦組織海馬CA1、CA2、CA3、CA4區(qū)神經(jīng)細(xì)胞磷酸化MEK1/2的表達(dá)情況,結(jié)果發(fā)現(xiàn),在NCI組磷酸化MEK1/2在各時間點均有表達(dá),但與Sham組相比無差異;DCI組在全腦缺血15分鐘、再灌注1、3、6小時各時間點腦組織海馬CA2區(qū)幾乎所有神經(jīng)細(xì)胞都發(fā)生了凋亡改變。采用MEK1/2的特異性阻斷劑PD98059后,海馬CA1、CA2、CA3和CA4各區(qū)神經(jīng)細(xì)胞磷酸化MEK1/2表達(dá)受到抑制;同時也可見使用PD98059后,能夠明顯減少DCI組全腦缺血15分鐘、再灌注1、3、6小時各時間點腦組織海馬CA1、CA2、CA3和CA4各區(qū)神經(jīng)細(xì)胞凋亡。(3)cPLA2免疫組化結(jié)果:DCI組全腦缺血15分鐘時,海馬CA1、CA2、CA3、CA4區(qū)中神經(jīng)細(xì)胞cPLA2的表達(dá)情況與Sham組和NCI組比較陽性表達(dá)顯著增加;而在再灌注3小時,cPLA2在海馬CA1、CA2、CA3、CA4區(qū)神經(jīng)細(xì)胞的表達(dá)結(jié)果顯示,DCI組比Sham組明顯增多。同時也發(fā)現(xiàn),在使用PD98059后,能夠減少糖尿病腦缺血全腦缺血15分鐘、再灌注1、3、6小時各時間點腦組織海馬CA1、CA2、CA3和CA4區(qū)cPLA2的表達(dá)。 結(jié)論(1)糖尿病高血糖能夠加重腦缺血再灌注時海馬CA1區(qū)、CA2區(qū)、CA3區(qū)、CA4區(qū)損傷,導(dǎo)致凋亡神經(jīng)細(xì)胞明顯增加。(2)糖尿病高血糖腦缺血時MEK和cPLA2激活表達(dá)顯著增加,可能與海馬各區(qū)神經(jīng)細(xì)胞凋亡增加有關(guān)。糖尿病高血糖能夠?qū)е翬RK1/2上游激酶MEK激活增加,使ERK1/2信號通路磷酸化上調(diào),通過激活下游作用底物cPLA2加重了腦缺血性損傷。
[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.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R363

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