EPO对新生大鼠缺氧缺血性脑损伤缺氧诱导因子1α及存活素表达的影响
发布时间：2018-10-13 09:04
【摘要】：目的：缺氧缺血性脑病(hypoxic-ischemic encephalopathy, HIE)是导致新生儿急性死亡及慢性神经系统损伤的主要原因之一。研究表明，促红细胞生成素(erythropoietin, EPO)有脑保护作用，本实验通过建立新生鼠缺氧缺血性脑损伤(hypoxic-ischemic brain damage, HIBD)模型，腹腔内注射外源性重组人促红细胞生成素(recombinant human erythropoietin, rhEPO)，观察EPO对新生鼠HIBD时缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)及存活素(Survivin)表达的影响，探讨其抗凋亡的可能机制，为临床应用EPO治疗新生儿HIE提供新的理论依据。 方法：将7d Wistar大鼠120只随机分为3组：假手术组(n=8)、HIBD组(n=56)、rhEPO治疗组(n=56)，后两者根据处死时间分为：3h组，6h组，12h组，24h组，48h组，3d组，7d组，每组8只。假手术组新生大鼠行常规消毒后切开颈部皮肤，分离左侧颈总动脉，不予缺氧处理；HIBD组新生大鼠分离左侧颈总动脉并给予结扎，后置于缺氧仓中（8%O2和92%N2混合气体）2h；rhEPO治疗组于HIBD模型建立后即刻、24h、48h连续腹腔注射rhEPO(5 000 IU/kg/d)。各组新生鼠于相应时间点处死取脑组织后观察脑外观的变化，采用HE染色镜下观察脑组织的病理学改变，应用免疫组化法测定HIF-1、Survivin的表达，用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法（terminal-deoxynucleotidyl transferasemediated nick end labeling, TUNEL）对细胞凋亡指数（apoptosis index, AI）进行检测。 结果：（1）新生大鼠缺氧缺血后可见大脑左侧有不同程度的水肿，rhEPO治疗后左侧脑水肿程度较HIBD组减轻。（2）HE染色：假手术组新生大鼠脑组织结构和细胞层次清楚，细胞排列规整，形态尚正常，可见少数细胞胞浆皱缩，核深染；HIBD组各时间点新生大鼠左侧脑组织呈现不同程度的水肿，可见坏死，胞核固缩、溶解，神经细胞数减少；rhEPO治疗组各时间点大鼠脑组织损伤程度与HIBD组相比有所减轻。（3）免疫组化染色：①HIF-1的表达：HIF-1阳性细胞主要表达于大脑皮层和海马区，阳性着色位于神经细胞胞质和（或）胞核，呈棕黄色细颗粒沉积。HIF-1的表达在假手术组中呈少量表达；在HIBD组，缺氧缺血后3h即增强，12h达高峰，后逐渐降低(P＜0.01)；rhEPO治疗组12h、24h、48h、3d的HIF-1表达水平较HIBD组明显减少(P＜0.05)。②Survivin的表达：Survivin阳性细胞主要表达于大脑皮层和海马区，阳性着色位于神经细胞胞质和（或）胞核，呈棕黄色细颗粒沉积。Survivin的表达在假手术组仅少量表达；在HIBD组，缺氧缺血后12h开始增高，7d明显增高，与假手术组相比，除3h、6h组，差异有统计学意义(P＜0.01)；rhEPO治疗组12h、24h、48h、3d的Survivin表达较HIBD组明显增高(P＜0.05)。（4）神经细胞凋亡趋势：细胞AI在假手术组中较低；在HIBD组缺氧缺血6h即增高，7d明显增高，与假手术组相比，除3h组，差异有统计学意义(P＜0.01)；rhEPO治疗组24h、48h、3d、7d的细胞AI值较HIBD组明显降低(P＜0.05)。 结论：(1)通过镜下观察HE染色结果证实，rhEPO治疗组各时间点与HIBD组比较，新生鼠脑组织病理损伤程度有所减轻，从病理学证实应用EPO治疗后可减轻HIBD。(2)脑缺氧缺血可导致脑皮质及海马区神经细胞凋亡，同时可提高HIF-1和Survivin的表达，说明HIF-1和Survivin共同参与了新生大鼠HIBD的病理过程。(3)应用EPO治疗后，通过抑制HIF-1的过表达及提高Survivin的表达，进而抑制神经细胞凋亡，从而在新生鼠HIBD时发挥其脑保护作用，这可能是EPO脑保护作用机制之一。
[Abstract]:Objective: The hypoxic ischemic encephalopathy (HIE) is one of the main causes of acute death and chronic nervous system injury in neonates. It is shown that erythropoietin (EPO) has the protective effect of brain. This experiment is to establish a model of hypoxic-ischemic brain injury (HIBD) in neonatal rats, and inject exogenous recombinant human erythropoietin (rhEPO) into the abdominal cavity. Objective To observe the effect of EPO on the expression of hypoxia-inducible factor-1 gene and survivin in neonatal rat HIBD, and to explore the possible mechanism of anti-apoptosis, and to provide a new theoretical basis for clinical application of EPO in the treatment of neonatal HIE. Methods: 120 Wistar rats were randomly divided into 3 groups: sham operation group (n = 8), HIBD group (n = 56), rhEPO treatment group (n = 56). The two groups were divided into three groups: 3h group, 6h group, 12h group, 24h group, 48h group, 3d group, 7d group, each group 8 The left carotid artery was isolated from the neonatal rats of HIBD group and ligated and placed in anoxic bin (8% O2 and 92% N2 mixed gas) 2h; rhEPO treatment group was established after the HIBD model was established. engraved, 24h, 48h continuous intraperitoneal injection of rhEPO (5,000 IU/ kg/ day The changes of brain appearance were observed after the rats were killed at corresponding time points, and the pathological changes of brain tissues were observed under HE staining. The expressions of survivin and Survivin were determined by immunohistochemistry. The apoptosis index (AI) was examined by TUNEL-mediated nick end labeling (TUNEL). Results: (1) After hypoxic ischemia in neonatal rats, there were different degrees of edema left on the left side of the brain, and the left brain edema after rhEPO treatment was higher than that of HIBD. (2) HE staining: The structure and cell level of brain tissue of neonatal rats in sham operation group are clear, the cell arrangement is regular, the morphology is still normal, few cell cytoplasm shrinkage and nuclear deep staining can be seen, and the left brain tissues of neonatal rats in the HIBD group show different degree of edema, which can See necrosis, cell nucleus contraction, dissolution, number of nerve cells decreased, and the degree of damage of brain tissue in rhEPO treatment group was compared with that of HIBD group. (3) Immunohistochemical staining: The expression of p27-1 was mainly expressed in the cerebral cortex and hippocampus, and the positive staining was located in the cytoplasm and/ or nucleus of the nerve cells. The expression of CD44v6-1 in the sham operation group was expressed in a small amount; in the HIBD group, after the hypoxia-ischemia 3h, the expression level was increased, the peak of 12h reached the peak, and then gradually decreased (P <0.01). The expression level of OPG-1 in rhEPO treatment group was significantly lower than that in HIBD group (P <0. 01). 05). Survivin expression: Survivin positive cells are mainly expressed in the cerebral cortex and hippocampus, and the positive staining is located in the cytoplasm and/ or nucleus of nerve cells. The expression of Survivin was only a small amount in sham operation group; in HIBD group, 12h after hypoxic ischemia increased, and 7d was significantly increased. Compared with sham operation group, the difference was significant (P <0.01); rhEPO treated group 12h, 24h. The expression of Survivin in 48h and 3d was higher than that in HIBD group (P <0.01). 05). (4) Neuronal apoptosis trend: The AI was lower in the sham operation group; in the HIBD group, the hypoxia ischemia 6h was increased, and 7d was significantly increased. Compared with the sham operation group, the difference was statistically significant (P <0.01). The rhEPO treatment group was 24h, 48h. The AI values of 3d and 7d were significantly lower than that in HIBD group (P <0.01). Conclusion: (1) Compared with HIBD group, the pathological damage degree of neonatal rat's brain tissue was decreased, and the pathological findings of rhEPO treated group decreased after EPO therapy. Mild HIBD. (2) Cerebral ischemia can lead to neuronal apoptosis in cerebral cortex and hippocampus, meanwhile, the expression of survivin-1 and Survivin can be improved, and the expression of survivin-1 and Survivin in neonatal rat HIBD is demonstrated. (3) After EPO is applied, the expression of survivin and the expression of Survivin are inhibited, so that the apoptosis of nerve cells can be inhibited, so that the brain protection effect can be exerted when the newborn mouse HIBD is HIBD, which may be EPO brain protection.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1
本文编号：2268035
[Abstract]:Objective: The hypoxic ischemic encephalopathy (HIE) is one of the main causes of acute death and chronic nervous system injury in neonates. It is shown that erythropoietin (EPO) has the protective effect of brain. This experiment is to establish a model of hypoxic-ischemic brain injury (HIBD) in neonatal rats, and inject exogenous recombinant human erythropoietin (rhEPO) into the abdominal cavity. Objective To observe the effect of EPO on the expression of hypoxia-inducible factor-1 gene and survivin in neonatal rat HIBD, and to explore the possible mechanism of anti-apoptosis, and to provide a new theoretical basis for clinical application of EPO in the treatment of neonatal HIE. Methods: 120 Wistar rats were randomly divided into 3 groups: sham operation group (n = 8), HIBD group (n = 56), rhEPO treatment group (n = 56). The two groups were divided into three groups: 3h group, 6h group, 12h group, 24h group, 48h group, 3d group, 7d group, each group 8 The left carotid artery was isolated from the neonatal rats of HIBD group and ligated and placed in anoxic bin (8% O2 and 92% N2 mixed gas) 2h; rhEPO treatment group was established after the HIBD model was established. engraved, 24h, 48h continuous intraperitoneal injection of rhEPO (5,000 IU/ kg/ day The changes of brain appearance were observed after the rats were killed at corresponding time points, and the pathological changes of brain tissues were observed under HE staining. The expressions of survivin and Survivin were determined by immunohistochemistry. The apoptosis index (AI) was examined by TUNEL-mediated nick end labeling (TUNEL). Results: (1) After hypoxic ischemia in neonatal rats, there were different degrees of edema left on the left side of the brain, and the left brain edema after rhEPO treatment was higher than that of HIBD. (2) HE staining: The structure and cell level of brain tissue of neonatal rats in sham operation group are clear, the cell arrangement is regular, the morphology is still normal, few cell cytoplasm shrinkage and nuclear deep staining can be seen, and the left brain tissues of neonatal rats in the HIBD group show different degree of edema, which can See necrosis, cell nucleus contraction, dissolution, number of nerve cells decreased, and the degree of damage of brain tissue in rhEPO treatment group was compared with that of HIBD group. (3) Immunohistochemical staining: The expression of p27-1 was mainly expressed in the cerebral cortex and hippocampus, and the positive staining was located in the cytoplasm and/ or nucleus of the nerve cells. The expression of CD44v6-1 in the sham operation group was expressed in a small amount; in the HIBD group, after the hypoxia-ischemia 3h, the expression level was increased, the peak of 12h reached the peak, and then gradually decreased (P <0.01). The expression level of OPG-1 in rhEPO treatment group was significantly lower than that in HIBD group (P <0. 01). 05). Survivin expression: Survivin positive cells are mainly expressed in the cerebral cortex and hippocampus, and the positive staining is located in the cytoplasm and/ or nucleus of nerve cells. The expression of Survivin was only a small amount in sham operation group; in HIBD group, 12h after hypoxic ischemia increased, and 7d was significantly increased. Compared with sham operation group, the difference was significant (P <0.01); rhEPO treated group 12h, 24h. The expression of Survivin in 48h and 3d was higher than that in HIBD group (P <0.01). 05). (4) Neuronal apoptosis trend: The AI was lower in the sham operation group; in the HIBD group, the hypoxia ischemia 6h was increased, and 7d was significantly increased. Compared with the sham operation group, the difference was statistically significant (P <0.01). The rhEPO treatment group was 24h, 48h. The AI values of 3d and 7d were significantly lower than that in HIBD group (P <0.01). Conclusion: (1) Compared with HIBD group, the pathological damage degree of neonatal rat's brain tissue was decreased, and the pathological findings of rhEPO treated group decreased after EPO therapy. Mild HIBD. (2) Cerebral ischemia can lead to neuronal apoptosis in cerebral cortex and hippocampus, meanwhile, the expression of survivin-1 and Survivin can be improved, and the expression of survivin-1 and Survivin in neonatal rat HIBD is demonstrated. (3) After EPO is applied, the expression of survivin and the expression of Survivin are inhibited, so that the apoptosis of nerve cells can be inhibited, so that the brain protection effect can be exerted when the newborn mouse HIBD is HIBD, which may be EPO brain protection.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R722.1
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