rhEPO对大鼠缺氧缺血性脑损伤后VEGF和VEGFR1表达的影响
发布时间：2018-08-09 15:11
【摘要】：背景 随着早产儿、低出生体重儿以及重度缺氧缺血性脑病(hypoxic-ischemic encephalopathy, HIE)新生儿存活率提高,新生儿脑损伤及其伴随的神经系统后遗症成为儿科领域关注的重要问题。研究证实脑室周围白质软化(Periventricular leukomalacia, PVL)是早产儿缺氧缺血性脑损伤主要病理学改变,引起国内外学者广泛的关注。近年来研究发现,促红细胞生成素不仅参与造血的调控,对神经系统也有保护作用,然而对于其作用机制尚未完全明了。重组人促红细胞生成素(recombinant human erythropoietin, rhEPO)是一种通过基因重组技术合成的糖蛋白,与天然EPO有着相同氨基酸序列,生物学活性也相似,在基础和临床研究中更显示了良好的疗效及应用前景。目的 检测外源性促红细胞生成素对缺氧缺血性脑损伤(hypoxic-ischemic brain damage, HIBD)新生大鼠脑组织中VEGF及受体表达的影响,探讨促红细胞生成素对脑损伤的保护机制,为临床缺氧缺血性脑损伤新生儿的早期治疗提供依据。方法 选择3日龄新生SD大鼠180只,随机分为5组：假手术组、对照组(模型组)、rhEPO小剂量治疗组(rhEPOS组)、rhEPO中剂量治疗组(rhEPOM组)、rhEPO大剂量治疗组(rhEPOH组),每组36只。制作缺氧缺血动物模型模拟早产儿脑室周围白质软化损害的病理改变。rhEPOS组、rhEPOM组、rhEPOH组在缺氧缺血后即刻分别给予1000U/kg.d,3000U/kg、5000U/kg.d的rhEPO腹腔注射,连续3天。对照组在术后于治疗组相同时间点给予腹腔注射等体积生理盐水。假手术组不予缺氧缺血处理也于相同时间点注射等体积生理盐水。各组均在每次注射后12h(即造模后12h、36h、60h)处死大鼠,手术取出大脑组织并保存备用。采用免疫组织化学技术及实时荧光定量PCR (Real-time Fluorescent Quantitative PCR, Real-Time PCR)技术,检测造模后12h、36h、60h血管内皮生长因子及血管内皮生长因子受体蛋白和基因的表达变化；分析rhEPO对大鼠缺氧缺血后VEGF和VEGFR1表达的影响。SPSS13.0进行统计分析,P0.05有统计学意义。结果1.神经行为学改变：对照组大鼠出现全身发绀,进乳少,活动少,反应差,频繁点头状抽搐,大小便失禁,部分动物于缺氧缺血过程中死亡。假手术组表现为进乳好,活动有力,精神反应好。rhEPOS组与对照组相比,行为学差别不大；rhEPOM组出现进乳尚可,活动较少,反应稍差；rhEPOH组出现进乳可,活动可自如,反应可。2.体重改变：在缺氧缺血后新生大鼠出现体重下降,体重增长缓慢情况,随着rhEPO用量的增大,动物的体重增长速度越接近正常生长速度。各组间使用单因素方差分析比较有统计学意义(P0.05)。3.脑组织病理形态学改变：假手术组脑白质细胞排列有序,细胞核圆形、淡染,层次分明,左右侧脑室对称。对照组在造模后出现脑白质区细胞肿胀、排列紊乱,胼胝体变薄,疏松坏死区域形成。在rhEPO干预下,治疗组脑组织病理学呈现较对照组明显好转,差异有统计学意义(P0.05)。4.免疫组化结果及Real-Time PCR结果:VEGF蛋白、VEGFR1蛋白在假手术组脑组织内呈低量表达,在对照组中呈高表达,差异有统计学意义(P0.05)。rhEPO治疗组中VEGF蛋白、VEGFR1蛋白的表达明显上升,与对照组相比,有统计学差异(P0.05),随治疗剂量的增加表达量依次增加,不同剂量之间有显著性差异(p0.05)。VEGFmRNA、VEGFR1mRNA在假手术组脑组织内呈低量表达,在对照组中呈高表达,差异有统计学意义(P0.05)。rhEPO治疗组中的VEGFmRNA、 VEGFR1mRNA表达明显上升,与对照组相比,有统计学差异(P0.05),随之治疗剂量的增加表达量依次增加,不同剂量之间有显著性差异(P0.05)。5.相关性分析：经Spearman秩相关性分析后,VEGF蛋白与VEGFR1蛋白两者的表达相关性分析显示二者呈正相关(r=0.881, P0.05); VEGFmRNA与VEGFR1mRNA两者的表达相关性分析显示二者呈正相关(r=0.854,P0.05)。结论1.rhEPO可显著增加缺氧缺血性脑损伤新生大鼠脑组织中VEGF、VEGFR1蛋白和基因的表达。2.缺氧缺血性脑损伤大鼠脑组织中VEGF、VEGFR1蛋白和基因的表达在一定范围内随rhEPO剂量的增加依次增加。3. rhEPO对缺氧缺血性脑损伤具有保护作用,作用机制可能是通过调节VEGF和VEGFR1系统起作用。
[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.
【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】：R722.1
，
本文编号：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.
【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】：R722.1
，
本文编号：2174484
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