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目的观察全脑缺血再灌注时,细胞外信号调节激酶(ERK)与无嘌呤/无嘧啶核酸内切酶/氧化还原因子-1(APE/Ref-1)蛋白在大鼠海马区神经元的表达。将跨细胞膜信号转导与胞内DNA损伤修复相联系,探讨ERK信号通路在大鼠全脑缺血再灌注中的作用。方法90只雄性SD大鼠随机分为3组:假手术组(S组)、全脑缺血再灌注组(IR组)、pd98059抑制剂组(PD组)。采用四血管阻断法建立大鼠全脑缺血再灌注模型,脑缺血时间5min,再灌注后2、6、12、24、48、72h,用免疫组化法分别检测磷酸化ERK和APE/Ref-1蛋白的表达,HE和TUNEL染色观察神经元凋亡。结果IR组CA1区TUNEL阳性细胞于缺血再灌注6h后开始出现,24h至48h达高峰;TUNEL阳性细胞在PD组表达最强。IR组磷酸化ERK于再灌注2h后,在海马CA3区可见明显表达,再灌注6h后逐渐下降,至再灌注后48h未见,PD组表达弱于IR组。IR组的APE/Ref-1蛋白于再灌注后2h在海马CA1区可见表达,2h到12h表达无明显变化,再灌注后24h明显减少,之后表达逐渐下降,PD组表达弱于IR组。磷酸化ERK表达下降的同时伴随有细胞凋亡的增加,APE/Ref-1在缺血再灌注后的表达变化与p-ERK一致,并且随着ERK磷酸化的被抑制APE/Ref-1的表达更加减弱。结论缺血再灌注中ERK通路的激活能增加APE/Ref-1蛋白的表达,对DNA的修复起支持作用,并在大鼠脑缺血再灌注时发挥抗神经元凋亡的保护作用。
Objective To observe the effect of extracellular signal-regulated kinase (ERK) and apurinic / apyrimidinic endonuclease / redox factor-1 (APE / Ref-1) protein in rat hippocampal neurons during global cerebral ischemia-reperfusion expression. The transmembrane signal transduction and intracellular DNA damage repair linked to explore the role of ERK signaling pathway in global cerebral ischemia-reperfusion in rats. Methods Ninety male Sprague-Dawley rats were randomly divided into three groups: sham operation group (S group), global cerebral ischemia reperfusion group (IR group) and PD98059 inhibitor group (PD group). A rat model of global cerebral ischemia / reperfusion was established by four-vessel occlusion. The cerebral ischemia time was 5 min, and at 2, 6, 12, 24, 48 and 72 h after reperfusion, the expressions of phosphorylated ERK and APE were detected by immunohistochemistry / Ref-1 protein expression, HE and TUNEL staining neuronal apoptosis. Results TUNEL-positive cells in CA1 area of IR group began to appear after 6h of ischemia-reperfusion and peaked from 24h to 48h. TUNEL-positive cells showed the strongest expression in PD group. ERK phosphorylated ERK in the hippocampal CA3 area 2h after reperfusion, and gradually decreased after 6h reperfusion, no expression 48h after reperfusion, PD group was weaker than the IR group. The expression of APE / Ref-1 protein in IR group was observed in hippocampal CA1 area 2h after reperfusion, but no significant change was seen in 2h to 12h. The expression of APE / Ref-1 protein in IR group decreased gradually after 24h. The expression of phosphorylated ERK decreased accompanied with the increase of apoptosis. The expression of APE / Ref-1 after ischemia-reperfusion was consistent with that of p-ERK, and inhibited with the phosphorylation of ERK APE / Ref-1 The expression is more diminished. Conclusion The activation of ERK pathway can increase the expression of APE / Ref-1 protein during ischemia-reperfusion, play a supportive role in the repair of DNA and play a protective role against neuronal apoptosis during cerebral ischemia-reperfusion in rats.