rhEPO對大鼠缺氧缺血性腦損傷后VEGF和VEGFR1表達的影響
發(fā)布時間：2018-08-09 15:11
【摘要】：背景 隨著早產兒、低出生體重兒以及重度缺氧缺血性腦病(hypoxic-ischemic encephalopathy, HIE)新生兒存活率提高,新生兒腦損傷及其伴隨的神經(jīng)系統(tǒng)后遺癥成為兒科領域關注的重要問題。研究證實腦室周圍白質軟化(Periventricular leukomalacia, PVL)是早產兒缺氧缺血性腦損傷主要病理學改變,引起國內外學者廣泛的關注。近年來研究發(fā)現(xiàn),促紅細胞生成素不僅參與造血的調控,對神經(jīng)系統(tǒng)也有保護作用,然而對于其作用機制尚未完全明了。重組人促紅細胞生成素(recombinant human erythropoietin, rhEPO)是一種通過基因重組技術合成的糖蛋白,與天然EPO有著相同氨基酸序列,生物學活性也相似,在基礎和臨床研究中更顯示了良好的療效及應用前景。目的 檢測外源性促紅細胞生成素對缺氧缺血性腦損傷(hypoxic-ischemic brain damage, HIBD)新生大鼠腦組織中VEGF及受體表達的影響,探討促紅細胞生成素對腦損傷的保護機制,為臨床缺氧缺血性腦損傷新生兒的早期治療提供依據(jù)。方法 選擇3日齡新生SD大鼠180只,隨機分為5組：假手術組、對照組(模型組)、rhEPO小劑量治療組(rhEPOS組)、rhEPO中劑量治療組(rhEPOM組)、rhEPO大劑量治療組(rhEPOH組),每組36只。制作缺氧缺血動物模型模擬早產兒腦室周圍白質軟化損害的病理改變。rhEPOS組、rhEPOM組、rhEPOH組在缺氧缺血后即刻分別給予1000U/kg.d,3000U/kg、5000U/kg.d的rhEPO腹腔注射,連續(xù)3天。對照組在術后于治療組相同時間點給予腹腔注射等體積生理鹽水。假手術組不予缺氧缺血處理也于相同時間點注射等體積生理鹽水。各組均在每次注射后12h(即造模后12h、36h、60h)處死大鼠,手術取出大腦組織并保存?zhèn)溆谩２捎妹庖呓M織化學技術及實時熒光定量PCR (Real-time Fluorescent Quantitative PCR, Real-Time PCR)技術,檢測造模后12h、36h、60h血管內皮生長因子及血管內皮生長因子受體蛋白和基因的表達變化；分析rhEPO對大鼠缺氧缺血后VEGF和VEGFR1表達的影響。SPSS13.0進行統(tǒng)計分析,P0.05有統(tǒng)計學意義。結果1.神經(jīng)行為學改變：對照組大鼠出現(xiàn)全身發(fā)紺,進乳少,活動少,反應差,頻繁點頭狀抽搐,大小便失禁,部分動物于缺氧缺血過程中死亡。假手術組表現(xiàn)為進乳好,活動有力,精神反應好。rhEPOS組與對照組相比,行為學差別不大；rhEPOM組出現(xiàn)進乳尚可,活動較少,反應稍差；rhEPOH組出現(xiàn)進乳可,活動可自如,反應可。2.體重改變：在缺氧缺血后新生大鼠出現(xiàn)體重下降,體重增長緩慢情況,隨著rhEPO用量的增大,動物的體重增長速度越接近正常生長速度。各組間使用單因素方差分析比較有統(tǒng)計學意義(P0.05)。3.腦組織病理形態(tài)學改變：假手術組腦白質細胞排列有序,細胞核圓形、淡染,層次分明,左右側腦室對稱。對照組在造模后出現(xiàn)腦白質區(qū)細胞腫脹、排列紊亂,胼胝體變薄,疏松壞死區(qū)域形成。在rhEPO干預下,治療組腦組織病理學呈現(xiàn)較對照組明顯好轉,差異有統(tǒng)計學意義(P0.05)。4.免疫組化結果及Real-Time PCR結果:VEGF蛋白、VEGFR1蛋白在假手術組腦組織內呈低量表達,在對照組中呈高表達,差異有統(tǒng)計學意義(P0.05)。rhEPO治療組中VEGF蛋白、VEGFR1蛋白的表達明顯上升,與對照組相比,有統(tǒng)計學差異(P0.05),隨治療劑量的增加表達量依次增加,不同劑量之間有顯著性差異(p0.05)。VEGFmRNA、VEGFR1mRNA在假手術組腦組織內呈低量表達,在對照組中呈高表達,差異有統(tǒng)計學意義(P0.05)。rhEPO治療組中的VEGFmRNA、 VEGFR1mRNA表達明顯上升,與對照組相比,有統(tǒng)計學差異(P0.05),隨之治療劑量的增加表達量依次增加,不同劑量之間有顯著性差異(P0.05)。5.相關性分析：經(jīng)Spearman秩相關性分析后,VEGF蛋白與VEGFR1蛋白兩者的表達相關性分析顯示二者呈正相關(r=0.881, P0.05); VEGFmRNA與VEGFR1mRNA兩者的表達相關性分析顯示二者呈正相關(r=0.854,P0.05)。結論1.rhEPO可顯著增加缺氧缺血性腦損傷新生大鼠腦組織中VEGF、VEGFR1蛋白和基因的表達。2.缺氧缺血性腦損傷大鼠腦組織中VEGF、VEGFR1蛋白和基因的表達在一定范圍內隨rhEPO劑量的增加依次增加。3. rhEPO對缺氧缺血性腦損傷具有保護作用,作用機制可能是通過調節(jié)VEGF和VEGFR1系統(tǒng)起作用。
[Abstract]:Background with premature infants, low birth weight infants and severe hypoxic ischemic encephalopathy (hypoxic-ischemic encephalopathy, HIE), the survival rate of newborns is increased. Neonatal brain injury and associated neurological sequelae have become an important issue in the pediatric field. The study confirmed that the periventricular softening (Periventricular leukomalacia, P) VL) is the main pathological change of hypoxic ischemic brain injury in preterm infants, which has aroused widespread concern at home and abroad. In recent years, it has been found that erythropoietin not only participates in the regulation of hematopoiesis, but also has protective effect on the nervous system, but the mechanism of its action is not completely clear. Recombinant human erythropoietin (recombinant human E) Rythropoietin, rhEPO) is a glycoprotein synthesized by gene recombination technology. It has the same amino acid sequence with natural EPO, and its biological activity is similar. It also shows good curative effect and application prospect in basic and clinical studies. Objective to detect exogenous erythropoietin for hypoxic ischemic brain injury (hypoxic-ischemic BRA). In damage, HIBD) the effect of VEGF and receptor expression in the brain tissue of newborn rats, explore the protective mechanism of erythropoietin on brain injury, provide the basis for the early treatment of neonatal hypoxic-ischemic brain injury. Methods 180 newborn SD rats of 3 days old were randomly divided into 5 groups: sham operation group, control group (model group), rhEPO small dose The dose treatment group (group rhEPOS), rhEPO medium dose treatment group (group rhEPOM), rhEPO large dose treatment group (group rhEPOH), 36 rats in each group. The histopathological changes in the hypoxic-ischemic animal model were made to simulate the pathological changes of the hypoxic damage around the ventricle of the preterm infants in the.RhEPOS group, the rhEPOM group, and the rhEPOH group were given 1000U/kg.d, 3000U/kg, 5000U/kg.d rhEP immediately after oxygen ischemia. O was injected intraperitoneally for 3 days. The control group was given equal volume of saline injected intraperitoneally at the same time point in the control group. The sham operation group had no hypoxic and ischemic treatment at the same time and injected the same volume of normal saline at the same time point. Each group died of 12h (12h, 36h, 60H) after each injection, and the brain tissue was removed and preserved. Using immunohistochemical technique and real-time fluorescent quantitative PCR (Real-time Fluorescent Quantitative PCR, Real-Time PCR), the expression changes of 12h, 36h, 60H vascular endothelial growth factor and vascular endothelial growth factor receptor protein and gene in 12h, 36h, 60H and rhEPO on rats after hypoxia and ischemia were analyzed. The.SPSS13.0 was statistically analyzed, and P0.05 had statistical significance. Results 1. neurobehavioral changes: the rats in the control group had cyanosis, less milk, less activity, poor response, frequent nod of convulsions, incontinence, and some animals died in the process of hypoxia and ischemia. The sham operation group showed good milk intake, strong activity, and good mental response to.RhEPOS Compared with the control group, there was little difference in behavior learning. Group rhEPOM appeared to be able to enter milk, less activity and less reaction; group rhEPOH appeared to be milk, the activity could be free, and the reaction could change the weight of.2. in the newborn rats after hypoxia ischemia, the weight increase of the newborn rats was slow, and the weight of the animals increased with the increase of rhEPO. The more close to normal growth rate, the comparison of the use of single factor analysis of variance in each group was statistically significant (P0.05).3. brain tissue pathological changes: the white matter cells in the sham operation group were arranged in order, the nucleus was round, the nucleus was pale, the level of the left and right ventricles were symmetrical. The pathological changes of brain tissue in the treatment group were obviously better than those of the control group under the intervention of rhEPO. The difference was statistically significant (P0.05).4. immunohistochemical results and Real-Time PCR results: VEGF protein, VEGFR1 protein was expressed in the sham operation group with low expression in the control group, and the difference was statistically significant. The expression of VEGF protein and VEGFR1 protein in the study group (P0.05).RhEPO was significantly higher than that in the control group (P0.05), with the increase in the expression of the therapeutic dose (P0.05). There was a significant difference (P0.05).VEGFmRNA between the different doses (P0.05) and the low expression of VEGFR1mRNA in the brain tissue of the sham operation group, which was higher in the control group. The expression of the difference was statistically significant (P0.05) in the.RhEPO treatment group, the expression of VEGFmRNA and VEGFR1mRNA increased obviously. Compared with the control group, there was a statistically significant difference (P0.05), and the increased expression of the treatment dose increased in turn, and there was a significant difference between different doses (P0.05).5. correlation analysis: after the Spearman rank correlation analysis, VEGF protein The correlation analysis with the expression of VEGFR1 protein showed positive correlation between the two (r=0.881, P0.05), and the correlation analysis between the expression of VEGFmRNA and VEGFR1mRNA showed that the two was positively correlated (r=0.854, P0.05). Conclusion 1.rhEPO can significantly increase the expression of VEGF, VEGFR1 protein and gene expression in the brain tissue of hypoxic and ischemic brain injury rats. The expression of VEGF, VEGFR1 protein and gene in brain tissue of rats with ischemic brain injury has a protective effect on hypoxic ischemic brain damage in a certain range with the increase of the dose of rhEPO, which may be mediated by the regulation of VEGF and VEGFR1 system.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R722.1
，
本文編號：2174484
[Abstract]:Background with premature infants, low birth weight infants and severe hypoxic ischemic encephalopathy (hypoxic-ischemic encephalopathy, HIE), the survival rate of newborns is increased. Neonatal brain injury and associated neurological sequelae have become an important issue in the pediatric field. The study confirmed that the periventricular softening (Periventricular leukomalacia, P) VL) is the main pathological change of hypoxic ischemic brain injury in preterm infants, which has aroused widespread concern at home and abroad. In recent years, it has been found that erythropoietin not only participates in the regulation of hematopoiesis, but also has protective effect on the nervous system, but the mechanism of its action is not completely clear. Recombinant human erythropoietin (recombinant human E) Rythropoietin, rhEPO) is a glycoprotein synthesized by gene recombination technology. It has the same amino acid sequence with natural EPO, and its biological activity is similar. It also shows good curative effect and application prospect in basic and clinical studies. Objective to detect exogenous erythropoietin for hypoxic ischemic brain injury (hypoxic-ischemic BRA). In damage, HIBD) the effect of VEGF and receptor expression in the brain tissue of newborn rats, explore the protective mechanism of erythropoietin on brain injury, provide the basis for the early treatment of neonatal hypoxic-ischemic brain injury. Methods 180 newborn SD rats of 3 days old were randomly divided into 5 groups: sham operation group, control group (model group), rhEPO small dose The dose treatment group (group rhEPOS), rhEPO medium dose treatment group (group rhEPOM), rhEPO large dose treatment group (group rhEPOH), 36 rats in each group. The histopathological changes in the hypoxic-ischemic animal model were made to simulate the pathological changes of the hypoxic damage around the ventricle of the preterm infants in the.RhEPOS group, the rhEPOM group, and the rhEPOH group were given 1000U/kg.d, 3000U/kg, 5000U/kg.d rhEP immediately after oxygen ischemia. O was injected intraperitoneally for 3 days. The control group was given equal volume of saline injected intraperitoneally at the same time point in the control group. The sham operation group had no hypoxic and ischemic treatment at the same time and injected the same volume of normal saline at the same time point. Each group died of 12h (12h, 36h, 60H) after each injection, and the brain tissue was removed and preserved. Using immunohistochemical technique and real-time fluorescent quantitative PCR (Real-time Fluorescent Quantitative PCR, Real-Time PCR), the expression changes of 12h, 36h, 60H vascular endothelial growth factor and vascular endothelial growth factor receptor protein and gene in 12h, 36h, 60H and rhEPO on rats after hypoxia and ischemia were analyzed. The.SPSS13.0 was statistically analyzed, and P0.05 had statistical significance. Results 1. neurobehavioral changes: the rats in the control group had cyanosis, less milk, less activity, poor response, frequent nod of convulsions, incontinence, and some animals died in the process of hypoxia and ischemia. The sham operation group showed good milk intake, strong activity, and good mental response to.RhEPOS Compared with the control group, there was little difference in behavior learning. Group rhEPOM appeared to be able to enter milk, less activity and less reaction; group rhEPOH appeared to be milk, the activity could be free, and the reaction could change the weight of.2. in the newborn rats after hypoxia ischemia, the weight increase of the newborn rats was slow, and the weight of the animals increased with the increase of rhEPO. The more close to normal growth rate, the comparison of the use of single factor analysis of variance in each group was statistically significant (P0.05).3. brain tissue pathological changes: the white matter cells in the sham operation group were arranged in order, the nucleus was round, the nucleus was pale, the level of the left and right ventricles were symmetrical. The pathological changes of brain tissue in the treatment group were obviously better than those of the control group under the intervention of rhEPO. The difference was statistically significant (P0.05).4. immunohistochemical results and Real-Time PCR results: VEGF protein, VEGFR1 protein was expressed in the sham operation group with low expression in the control group, and the difference was statistically significant. The expression of VEGF protein and VEGFR1 protein in the study group (P0.05).RhEPO was significantly higher than that in the control group (P0.05), with the increase in the expression of the therapeutic dose (P0.05). There was a significant difference (P0.05).VEGFmRNA between the different doses (P0.05) and the low expression of VEGFR1mRNA in the brain tissue of the sham operation group, which was higher in the control group. The expression of the difference was statistically significant (P0.05) in the.RhEPO treatment group, the expression of VEGFmRNA and VEGFR1mRNA increased obviously. Compared with the control group, there was a statistically significant difference (P0.05), and the increased expression of the treatment dose increased in turn, and there was a significant difference between different doses (P0.05).5. correlation analysis: after the Spearman rank correlation analysis, VEGF protein The correlation analysis with the expression of VEGFR1 protein showed positive correlation between the two (r=0.881, P0.05), and the correlation analysis between the expression of VEGFmRNA and VEGFR1mRNA showed that the two was positively correlated (r=0.854, P0.05). Conclusion 1.rhEPO can significantly increase the expression of VEGF, VEGFR1 protein and gene expression in the brain tissue of hypoxic and ischemic brain injury rats. The expression of VEGF, VEGFR1 protein and gene in brain tissue of rats with ischemic brain injury has a protective effect on hypoxic ischemic brain damage in a certain range with the increase of the dose of rhEPO, which may be mediated by the regulation of VEGF and VEGFR1 system.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R722.1
，
本文編號：2174484
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