促红细胞生成素对脑缺血再灌注损伤大鼠脑Cdk5表达的影响

发布时间：2018-06-22 07:42

  本文选题：促红细胞生成素 + Cdk5　； 参考：《新乡医学院》2014年硕士论文

【摘要】：背景脑缺血是导致老年人死亡和残疾最常见的一种疾病,目前其治疗效果仍不尽人意。近年来研究发现促红细胞生成素(Erythropoietin,EPO)对脑缺血再灌注损伤(cerebral ischemia/reperfusion injury)有一定的保护作用,但EPO对大鼠脑缺血再灌注损伤脑组织周期蛋白依赖性蛋白激酶5(cyclin-dependent kinase 5, Cdk5)的表达是否有影响；国内外尚未见报道。目的通过研究EPO对Sprague-Dawley (SD)大鼠脑缺血再灌注损伤脑组织中Cdk5表达的影响,探讨EPO在脑缺血再灌注损伤中的保护作用机制。方法 将72只雄性SD大鼠随机分成四组：正常组、假手术组、缺血再灌注(ischemia-reperfusion, I/R)组和EPO+I/R组。采用Zea Longa线栓法建立大鼠左侧大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,EPO+I/R组于脑缺血时腹腔注射EPO3000 IU/kg, I/R组、假手术组和正常组于相应的时间点腹腔注射等量生理盐水,I/R组和EPO+I/R组于缺血2h拔出栓线实施再灌注,四组均于缺血2h再灌注24h后对动物进行脑损伤评估,检测各组大鼠死亡率、Longa评分标准进行神经功能缺陷评分以及2,3,5-三苯基氯化四氮唑(2,3,5-triphenyl four azole nitrogen chloride, TTC)染色测量脑梗死体积；采用免疫荧光和免疫印迹(Western blotting)方法检测大鼠大脑皮质和海马组织中Cdk5蛋白的表达。结果1.EPO对脑缺血再灌注损伤大鼠死亡率、神经功能缺陷评分的影响脑缺血2h再灌注24h, EPO+I/R组大鼠死亡率(21.74%)明显低于I/R组(33.33%),且EPO+I/R组大鼠神经功能缺陷评分(1.94+0.73)也显著低于I/R组(2.50±0.52)(P0.05)；正常组和假手术组无动物死亡,神经功能缺陷评分均为0分。2.EPO对脑缺血再灌注损伤大鼠脑梗死体积的影响TTC染色结果显示,正常组和假手术组两侧大脑半球呈均匀红色,I/R组和EPO+I/R组正常脑组织染为红色,左侧脑梗死组织失染呈苍白色,且与I/R组(36.38%±3.30%)相比,EPO+I/R组脑梗死体积(21.88%±2.96%)显著减小(P0.05)。3.EPO对脑缺血再灌注损伤大鼠大脑皮质Cdk5表达的影响(1)免疫荧光结果显示,各组大鼠大脑皮质均可见Cdk5免疫阳性细胞,阳性细胞胞膜和胞质呈红色,与正常组(10.83+3.06)相比,假手术组左侧脑皮质Cdk5阳性表达细胞数(11.33±3.50)无显著性差异；与假手术组(11.33±3.50)相比,I/R组左侧脑皮质Cdk5阳性表达细胞数(40.33±5.34)显著增多(P0.05)；与I/R组(40.33±5.34)相比,EPO+I/R组左侧脑皮质Cdk5阳性表达细胞数(24.17±5.81)显著降低(P0.05)。(2)Western blotting电泳条带灰度分析结果显示,与正常组(0.502±0.093)相比,假手术组左侧脑皮质中Cdk5蛋白表达量(0.540±0.099)差异无显著性；与假手术组(0.540±0.099)相比,I/R组左侧脑皮质中Cdk5蛋白表达量(1.227+0.115)显著增多(P0.05)；与I/R组(1.227±0.115)相比,EPO+I/R组左侧脑皮质中Cdk5蛋白表达量(0.592±0.113)显著降低(P0.05)。4.EPO对脑缺血再灌注损伤大鼠海马Cdk5表达的影响(1)免疫荧光结果显示,各组大鼠海马组织中均可见Cdk5免疫阳性细胞,阳性细胞胞膜及胞质呈红色,且与正常组(9.33±2.80)相比,假手术组左侧海马CA1区Cdk5阳性表达细胞数(9.50±1.87)无显著性差异；与假手术组(9.50±1.87)相比,I/R组左侧海马CA1区Cdk5阳性表达细胞数(31.33±6.62)显著增多(P0.05)；与I/R组(31.33±6.62)相比,EPO+I/R组左侧海马CA1区Cdk5阳性表达细胞数(13.17±3.31)显著降低(P0.05)。(2)Western blotting电泳条带灰度分析结果显示,与正常组(0.410±0.067)相比,假手术组左侧海马组织中Cdk5蛋白表达量(0.390±0.085)差异无显著性；与假手术组(0.390-±0.085)相比,I/R组左侧海马组织中Cdk5蛋白表达量(1.228±0.074)显著增多(P0.05)；与I/R组(1.228±0.074)相比,EPO+I/R组左侧海马组织中Cdk5蛋白表达量(0.890±0.079)显著降低(P0.05)。结论1.EPO可减小大鼠脑缺血再灌注损伤后脑梗死体积,改善神经功能缺陷,具有神经保护作用。2.大鼠脑缺血再灌注损伤后Cdk5蛋白表达上调,EPO可抑制脑缺血再灌注损伤大鼠Cdk5蛋白的表达,这可能是其发挥神经保护作用的机制之一。
[Abstract]:Background cerebral ischemia is the most common disease causing death and disability in the elderly, and its therapeutic effect is still unsatisfactory. In recent years, it has been found that Erythropoietin (EPO) has a definite protective effect on cerebral ischemia-reperfusion injury (cerebral ischemia/reperfusion injury), but EPO on cerebral ischemia reperfusion injury in rats The effect of the expression of cyclin dependent protein kinase 5 (cyclin-dependent kinase 5, Cdk5) on the cyclin dependent brain tissue is not reported at home and abroad. The purpose of this study was to investigate the effect of EPO on the expression of Cdk5 in cerebral ischemia reperfusion injury in Sprague-Dawley (SD) rats, and to explore the protective mechanism of EPO in cerebral ischemia-reperfusion injury. Methods 72 male SD rats were randomly divided into four groups: normal group, sham operation group, ischemia reperfusion (ischemia-reperfusion, I/R) group and EPO+I/R group. The left middle cerebral artery embolism (middle cerebral artery occlusion, MCAO) model was established by Zea Longa thread embolus. The sham operation group and the normal group were intraperitoneally injected with equal amount of saline at the corresponding time points, the I/R group and the EPO+I/R group were pulled out of the ischemic 2H to carry out the reperfusion. The four groups were assessed the brain damage of the animals after the ischemia 2H reperfusion 24h, and the mortality of the rats in each group was detected. The Longa score was marked by the neurological deficit score and the 2,3,5- three phenyl group. The volume of cerebral infarction was measured by 2,3,5-triphenyl four azole nitrogen chloride (TTC) staining, and the expression of Cdk5 protein in the cerebral cortex and hippocampus of rats was detected by immunofluorescence and immunoblotting (Western blotting). Results 1.EPO on the mortality of rats injured by cerebral ischemia and reperfusion and the shadow of neurological deficit score Rounded cerebral ischemia 2H reperfusion 24h, the mortality rate of rats in group EPO+I/R (21.74%) was significantly lower than that in group I/R (33.33%), and the nerve function defect score (1.94+0.73) in group EPO+I/R was significantly lower than that in group I/R (2.50 + 0.52) (P0.05), and there was no animal death in the normal group and sham operation group, and the score of the function defect of the deity was 0.2.EPO to the cerebral ischemia reperfusion injury rats The effect of TTC staining on the volume of cerebral infarction showed that the cerebral hemispheres on both sides of the normal group and the sham operation group were red, the normal brain tissue in the I/R group and the EPO+I/R group were red, the left cerebral infarction tissue was pale, and compared with the I/R group (36.38% + 3.30%), the dead volume of the EPO+I/R group (21.88% + 2.96%) decreased significantly (P0.05).3.EPO to brain deficiency. The effect of blood reperfusion injury on the expression of Cdk5 in the cerebral cortex of rats (1) immunofluorescence showed that Cdk5 positive cells were found in the cerebral cortex of rats in all groups, and the cell membrane and cytoplasm of the positive cells were red. Compared with the normal group (10.83+3.06), there was no significant difference in the number of Cdk5 positive cells in the left cerebral cortex of the sham operation group (11.33 + 3.50). Compared with the operation group (11.33 + 3.50), the number of Cdk5 positive cells in the left cerebral cortex of the I/R group (40.33 + 5.34) increased significantly (P0.05). Compared with the I/R group (40.33 + 5.34), the number of Cdk5 positive cells in the left cerebral cortex of EPO+I/R group (24.17 + 5.81) was significantly decreased (P0.05). (2) the results of the Western blotting electrophoresis strip gray analysis showed that the group (0.502 + 0) was compared with the normal group (0.502 + 3.50). .093) there was no significant difference in the expression of Cdk5 protein in the left cerebral cortex of the sham operation group (0.540 + 0.099). Compared with the sham group (0.540 + 0.099), the Cdk5 protein expression (1.227+0.115) in the left cerebral cortex of the I/R group increased significantly (P0.05), and the Cdk5 protein expression in the left cerebral cortex of EPO+I/R group was 0.592 + 0.11 compared with the I/R group (1.227 + 0.115). 3) the effect of (P0.05).4.EPO on the expression of Cdk5 in hippocampus of rats with cerebral ischemia-reperfusion injury (1) immunofluorescence showed that Cdk5 immunoreactive cells were found in the hippocampus of all rats, and the cell membrane and cytoplasm of the positive cells were red, and compared with the normal group (9.33 + 2.80), the number of Cdk5 positive cells in the left hippocampus CA1 region of the sham operation group was compared with that of the normal group (9.33 + 2.80). There was no significant difference in 9.50 + 1.87. Compared with the sham operation group (9.50 + 1.87), the number of Cdk5 positive cells in the left hippocampal CA1 region of the I/R group increased significantly (P0.05). Compared with the I/R group (31.33 + 6.62), the number of Cdk5 positive cells in the CA1 region of the left hippocampus of EPO+I/R group (13.17 + 3.31) was significantly decreased (P0.05). (2) Western blotting electrophoresis strips with ash Compared with the normal group (0.410 + 0.067), there was no significant difference in the expression of Cdk5 protein (0.390 + 0.085) in the left hippocampus of the sham operation group. Compared with the sham group (0.390- + 0.085), the expression of Cdk5 protein in the left hippocampus of the I/R group was significantly increased (P0.05), and compared with the I/R group (1.228 + 0.074), EPO+I/R The expression of Cdk5 protein in the left hippocampus of the group (0.890 + 0.079) decreased significantly (P0.05). Conclusion 1.EPO can reduce the volume of cerebral infarction after cerebral ischemia-reperfusion injury in rats and improve the neural function defects. The expression of Cdk5 protein is up regulation in.2. rats after cerebral ischemia-reperfusion injury, and EPO can inhibit Cdk in rats with cerebral ischemia reperfusion injury. 5 the expression of protein may be one of the mechanisms of its neuroprotective effect.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3

【参考文献】

相关期刊论文 前2条

1 刘慧丽;梁丽贞;吴春丽;;促红细胞生成素和血管紧张素受体拮抗剂对脑缺血再灌注后梗死体积和脑组织水肿的影响[J];实用药物与临床;2014年03期

2 章军建,阮旭中,张苏明,王伟;大鼠局灶性脑缺血再灌流半暗带神经细胞坏死与凋亡的动态变化[J];中华老年医学杂志;1999年03期

，

本文编号：2052114