本文選題：促紅細(xì)胞生成素 + Cdk5��； 參考：《新鄉(xiāng)醫(yī)學(xué)院》2014年碩士論文

【摘要】：背景腦缺血是導(dǎo)致老年人死亡和殘疾最常見的一種疾病,目前其治療效果仍不盡人意。近年來研究發(fā)現(xiàn)促紅細(xì)胞生成素(Erythropoietin,EPO)對腦缺血再灌注損傷(cerebral ischemia/reperfusion injury)有一定的保護(hù)作用,但EPO對大鼠腦缺血再灌注損傷腦組織周期蛋白依賴性蛋白激酶5(cyclin-dependent kinase 5, Cdk5)的表達(dá)是否有影響；國內(nèi)外尚未見報(bào)道。目的通過研究EPO對Sprague-Dawley (SD)大鼠腦缺血再灌注損傷腦組織中Cdk5表達(dá)的影響,探討EPO在腦缺血再灌注損傷中的保護(hù)作用機(jī)制。方法 將72只雄性SD大鼠隨機(jī)分成四組：正常組、假手術(shù)組、缺血再灌注(ischemia-reperfusion, I/R)組和EPO+I/R組。采用Zea Longa線栓法建立大鼠左側(cè)大腦中動脈栓塞(middle cerebral artery occlusion,MCAO)模型,EPO+I/R組于腦缺血時(shí)腹腔注射EPO3000 IU/kg, I/R組、假手術(shù)組和正常組于相應(yīng)的時(shí)間點(diǎn)腹腔注射等量生理鹽水,I/R組和EPO+I/R組于缺血2h拔出栓線實(shí)施再灌注,四組均于缺血2h再灌注24h后對動物進(jìn)行腦損傷評估,檢測各組大鼠死亡率、Longa評分標(biāo)準(zhǔn)進(jìn)行神經(jīng)功能缺陷評分以及2,3,5-三苯基氯化四氮唑(2,3,5-triphenyl four azole nitrogen chloride, TTC)染色測量腦梗死體積；采用免疫熒光和免疫印跡(Western blotting)方法檢測大鼠大腦皮質(zhì)和海馬組織中Cdk5蛋白的表達(dá)。結(jié)果1.EPO對腦缺血再灌注損傷大鼠死亡率、神經(jīng)功能缺陷評分的影響腦缺血2h再灌注24h, EPO+I/R組大鼠死亡率(21.74%)明顯低于I/R組(33.33%),且EPO+I/R組大鼠神經(jīng)功能缺陷評分(1.94+0.73)也顯著低于I/R組(2.50±0.52)(P0.05)；正常組和假手術(shù)組無動物死亡,神經(jīng)功能缺陷評分均為0分。2.EPO對腦缺血再灌注損傷大鼠腦梗死體積的影響TTC染色結(jié)果顯示,正常組和假手術(shù)組兩側(cè)大腦半球呈均勻紅色,I/R組和EPO+I/R組正常腦組織染為紅色,左側(cè)腦梗死組織失染呈蒼白色,且與I/R組(36.38%±3.30%)相比,EPO+I/R組腦梗死體積(21.88%±2.96%)顯著減小(P0.05)。3.EPO對腦缺血再灌注損傷大鼠大腦皮質(zhì)Cdk5表達(dá)的影響(1)免疫熒光結(jié)果顯示,各組大鼠大腦皮質(zhì)均可見Cdk5免疫陽性細(xì)胞,陽性細(xì)胞胞膜和胞質(zhì)呈紅色,與正常組(10.83+3.06)相比,假手術(shù)組左側(cè)腦皮質(zhì)Cdk5陽性表達(dá)細(xì)胞數(shù)(11.33±3.50)無顯著性差異；與假手術(shù)組(11.33±3.50)相比,I/R組左側(cè)腦皮質(zhì)Cdk5陽性表達(dá)細(xì)胞數(shù)(40.33±5.34)顯著增多(P0.05)；與I/R組(40.33±5.34)相比,EPO+I/R組左側(cè)腦皮質(zhì)Cdk5陽性表達(dá)細(xì)胞數(shù)(24.17±5.81)顯著降低(P0.05)。(2)Western blotting電泳條帶灰度分析結(jié)果顯示,與正常組(0.502±0.093)相比,假手術(shù)組左側(cè)腦皮質(zhì)中Cdk5蛋白表達(dá)量(0.540±0.099)差異無顯著性；與假手術(shù)組(0.540±0.099)相比,I/R組左側(cè)腦皮質(zhì)中Cdk5蛋白表達(dá)量(1.227+0.115)顯著增多(P0.05)；與I/R組(1.227±0.115)相比,EPO+I/R組左側(cè)腦皮質(zhì)中Cdk5蛋白表達(dá)量(0.592±0.113)顯著降低(P0.05)。4.EPO對腦缺血再灌注損傷大鼠海馬Cdk5表達(dá)的影響(1)免疫熒光結(jié)果顯示,各組大鼠海馬組織中均可見Cdk5免疫陽性細(xì)胞,陽性細(xì)胞胞膜及胞質(zhì)呈紅色,且與正常組(9.33±2.80)相比,假手術(shù)組左側(cè)海馬CA1區(qū)Cdk5陽性表達(dá)細(xì)胞數(shù)(9.50±1.87)無顯著性差異；與假手術(shù)組(9.50±1.87)相比,I/R組左側(cè)海馬CA1區(qū)Cdk5陽性表達(dá)細(xì)胞數(shù)(31.33±6.62)顯著增多(P0.05)；與I/R組(31.33±6.62)相比,EPO+I/R組左側(cè)海馬CA1區(qū)Cdk5陽性表達(dá)細(xì)胞數(shù)(13.17±3.31)顯著降低(P0.05)。(2)Western blotting電泳條帶灰度分析結(jié)果顯示,與正常組(0.410±0.067)相比,假手術(shù)組左側(cè)海馬組織中Cdk5蛋白表達(dá)量(0.390±0.085)差異無顯著性；與假手術(shù)組(0.390-±0.085)相比,I/R組左側(cè)海馬組織中Cdk5蛋白表達(dá)量(1.228±0.074)顯著增多(P0.05)；與I/R組(1.228±0.074)相比,EPO+I/R組左側(cè)海馬組織中Cdk5蛋白表達(dá)量(0.890±0.079)顯著降低(P0.05)。結(jié)論1.EPO可減小大鼠腦缺血再灌注損傷后腦梗死體積,改善神經(jīng)功能缺陷,具有神經(jīng)保護(hù)作用。2.大鼠腦缺血再灌注損傷后Cdk5蛋白表達(dá)上調(diào),EPO可抑制腦缺血再灌注損傷大鼠Cdk5蛋白的表達(dá),這可能是其發(fā)揮神經(jīng)保護(hù)作用的機(jī)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.
【學(xué)位授予單位】：新鄉(xiāng)醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3

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本文編號：2052114