EPO对新生大鼠缺氧缺血性脑损伤时脑组织Caspase-9及spase-3的影响

EPO对新生大鼠缺氧缺血性脑损伤时脑组织Caspase-9和Caspase-3的影响研究生：白丹专业：儿科学导师：阴怀清摘要目的：新生儿缺氧缺血性脑损伤(hypoxic-ischemic encephalopathy, HIBD)是由围生期窒息所引起的，常危及新生儿生命，也是导致儿童神经系统损伤的常见原因之一。

近年来研究发现，促红细胞生成素(erythropoietin, EPO)对缺氧缺血性脑损伤时的脑组织有保护作用，本实验通过建立新生大鼠缺氧缺血性脑损伤(HIBD)模型，腹腔注射外源性重组人促红细胞生成素(recombinant human erythropoietin, rhEPO)，观察新生大鼠HIBD后脑组织病理变化，并观察EPO 对新生大鼠HIBD时脑组织Caspase-9和Caspase-3表达的影响，探讨EPO对新生大鼠HIBD 时脑组织产生的保护作用及产生这种保护作用的可能机制，以期为临床治疗新生儿HIBD 提供有效的治疗方法。

方法：(1)制备新生大鼠HIBD模型。

(2)将新生7日龄Wistar大鼠120只随机分为3组：假手术组，缺氧缺血组(HIBD组)，rhEPO治疗组，每组各40只，各组根据不同时间点又分成5个亚组：6h组，12h组，24h组，48h组，72h组，每亚组8只。

假手术组给予颈正中切口游离左侧颈总动脉，不进行缺氧缺血；HIBD组游离并结扎左侧颈总动脉后置于低氧环境中2h； rhEPO治疗组于缺氧缺血后立即腹腔注射rhEPO(5000U/kg/d)。

各组新生大鼠于相应时间点断头取脑组织后肉眼观察脑组织大体形态的改变，石蜡切片行HE染色光镜下观察左侧脑组织病理变化，免疫组织化学方法检测三组大鼠不同时间点左侧脑组织Caspase-9和Caspase-3表达。

结果：(1)缺氧缺血后可见左侧脑组织大体有不同程度的水肿，rhEPO治疗后水肿在一定程度上有所减轻。

(2)HE染色：假手术组脑组织结构及细胞层次清晰，神经元排列整齐紧密，仅见个别细胞体积缩小，核深染；HIBD组各时间点出现不同程度的神经细胞肿胀，变性，坏死，细胞核碎裂，溶解，神经元数目减少，胶质细胞增生；rhEPO治疗组各时间点损伤程度较HIBD组有所减轻，神经细胞存活数量多，细胞排列尚规则，仅见少量神经细胞变性坏死。

(3)免疫组织化学染色：①Caspase-9表达：Caspase-9阳性染色呈棕黄色的细颗粒沉积，阳性细胞广泛表达于大脑皮层和海马，阳性着色于神经元胞浆和胞核内均可见，在假手术组呈低水平表达，各时间点无明显变化；HIBD组Caspase-9的表达在缺氧缺血6h 即增强，12h时逐渐升高，24～72h维持在高峰水平（P<0.05）；rhEPO治疗组各时间点Caspase-9阳性颗粒染色较HIBD组染色浅，数量有所下降，阳性细胞平均灰度值较HIBD组高，差异均有统计学意义(all P<0.01)。

②Caspase-3表达：Caspase-3阳性染色呈棕黄色细颗粒沉积，阳性细胞广泛表达于大脑皮层和海马，阳性着色于神经元胞浆和胞核内均可见，Caspase-3在假手术组低水平表达；HIBD组Caspase-3的表达在缺氧缺血6h即增强，12h时逐渐升高，24～48h达高峰，72h稍有降低（P<0.05）；rhEPO治疗组各时间点Caspase-3阳性颗粒染色较HIBD组染色浅，数量有所下降，阳性细胞平均灰度值较HIBD组高，差异均有统计学意义(all P<0.01)。

结论：(1) 通过对缺氧缺血后新生大鼠脑组织大体观及病理学观察，证实新生大鼠缺氧缺血性脑损伤模型建立成功，HIBD组Caspase-9和Caspase-3的表达较假手术组有所增加，说明Caspase-9和Caspase-3参与了新生大鼠脑组织HIBD的发生发展过程。

(2)rhEPO可下调Caspase-9和Caspase-3的表达，从而发挥对缺氧缺血性脑损伤时脑组织的保护作用，为临床治疗新生儿缺氧缺血性脑损伤提供新的途径。

关键词：EPO；HIBD；新生大鼠；Caspase-9；Caspase-3Effect of exogenous erythropoietin on Caspase-9 and Caspase-3expression in newborn rats after hypoxia-ischemia brain damageAbstractObjective: Neonatal Hypoxic-Ischemic Brain Damage (HIBD) is caused by perinatal asphyxia often life-threatening newborns, it is also a common cause of children nervous system damage. Recent study has found that EPO (erythropoietin, EPO) has a protective effect on the hypoxic-ischemic injured brain tissue. This experiment by creating neonatal rats hypoxic-ischemic brain damage (HIBD) model. Intraperitoneal injection of exogenous recombinant human erythropoietin to observe the pathological change of brain tissue in HIBD neonatal rats and the effects of exogenous erythropoietin (EPO) on expression of Caspase-9 and Caspase-3，so as to explore the possible mechanism of the neuroprotective effect of EPO in HIBD neonatal rats, our experiment may provide an effective treatment for clinical therapy of neonatal HIBD.Methods: (1) Preparation of neonatal rat HIBD model. (2)A total of 120 seven-day-old neonatal rats were randomly divided into three groups: sham-operated group, HIBD group and rhEPO-treated group. Each group was further divided into five sub-groups (n=8 each) based on different time points after HIBD (6h, 12h, 24h, 48h, 72h) respectively. In sham-operated group，given median neck incision and free left common carotid artery didn’t hypoxic-ischemic. In HIBD group free and ligate left common carotid artery, then given hypoxic 2h. In rhEPO-treated group, immediately give a single intraperitoneal injection of rhEPO(5000U/kg/d) after hypoxic-ischemic brain damage. Observed the morphological changes in brain tissue after each group of neonatal rats were decapitation and collected the brain tissue at a corresponding time points. Observed the left side of brain tissue paraffin sections under the HE dyed light microscope to determine the pathological changes. Immunohistochemical technique was used to determine the expression of Caspase-9 and Caspase-3 in brain tissues.Results: (1) Varying degrees of edema generally can be seen on the left brain tissue after HIBD, rhEPO treatment reduced certain extent of edema. (2)HE dyed: In sham-group, brain structure and the cellular level are clear, neurons arranged closely, only see the individual cell volume shrinkage, nucleus deeply stained. HIBD group at different time points with varying degrees of nerve cell swelling, degeneration, necrosis, nuclear fragmentation, dissolution, reduction in the number of neurons, neuroglial cell proliferation. rhEPO treatment group compared to the HIBD group, the degree of injury was reduced, most of nerve cell survived, cell arrangement still in rules, only see a small amount of nerve cell degeneration and necrosis. (3)Immunohistochemical①-9 expression: Caspase-9 positive stained was brown fine particle staining results: Caspasedeposition, the positive cells are widely expressed in the cerebral cortex and hippocampus, positive staining in the cytoplasm and nuclei of neurons were seen, in the sham group showed a low level of expression, no significant changes at each time point; The expression of Caspase-9 in HIBD group increased at 6h after HIBD, 12h gradually increased, 24h～72h maintain the peak level（P<0.05); in rhEPO-treatment group at each time points Caspase-9 positive stained particle compared to the HIBD group were shallow, numbers were reduced, average gray value②-3 expression: higher than HIBD group, the difference was significant (all p<0.01). CaspaseCaspase-3 positive staining showed brown fine particle deposition, the positive cells are widely expressed in the cerebral cortex and hippocampus, positive staining in the cytoplasm and nuclei of neurons were seen, Caspase-3 Expression in the sham group at the low level; The expression of Caspase-3 in HIBD group increased at 6h after HIBD also, 12h gradually increased, the peak reached on 24h～48h and 72h gradually decreased（P<0.05); in rhEPO-treatment group at each time points Caspase-3 positive stained particle compared to the HIBD group were shallow, numbers were reduced, average gray value higher than HIBD group, the difference was significant (all p<0.01).Conclusion: (1)Through the general concept and pathological observation of the HIBD neonatal rat brain tissue, confirmed that HIBD model is established successfully, HIBD group of Caspase-9 and Caspase-3 expression compared with the sham-operated group was increased, indicating that Caspase-9 and Caspase-3 participate in the occurrence and development process of HIBD of neonatal rat. (2) rhEPO reduce the expression of Caspase-9 and Caspase-3, which can protect the HIBD brain tissue, for clinical treatment of neonatal hypoxic-ischemic brain injury provide a new way.Key words: EPO；HIBD；neonatal rats；Caspase-9；Caspase-3缩 略 词 简 表缩写英文全称中文全称EPO erythropoietin 促红细胞生成素receptor 促红细胞生成素受体EPO-R erythropoietinrhEPO recombinant human erythropoietin 重组人促红细胞生成素HIBD hypoxic-ischemic brain damage 缺氧缺血性脑损伤HIE hypoxic-ischemic encephalopathy 缺氧缺血性脑病Caspase-9 Caspase-9 半胱氨酸天冬氨酸蛋白酶-9 Casapse-3 Caspase-3 半胱氨酸天冬氨酸蛋白酶-3 DAB diaminobenzidine 二氨基联苯胺HE hematoxylin-eosin 苏木精-伊红PBS phosphate buffer saline 磷酸盐缓冲液PCD programmed cell death 细胞程序性死亡Apaf-1 apoptosis protease activating 1 凋亡蛋白激活因子-1cyt-c cyt-c 细胞色素酶cMPTP methyl phenyl tetrahydropyridine 线粒体通透性运输孔道Fas Fas 细胞表面死亡受体 PARP Polypolymerase ADP核糖聚合酶ADP-ribosekinase 局部粘着斑激酶FAK Focaladhesion山西医科大学硕士学位论文学位论文独创性声明本人声明，所呈交的学位论文系在导师指导下本文独立完成的研究成果。