重组人促红细胞生成素对脑出血后神经保护作用及机制研究

发布时间：2018-04-17 05:12

本文选题：重组人促红细胞生成素 + 脑出血　；参考：《青岛大学》2017年硕士论文

【摘要】：背景:随着人口老龄化程度的加速,脑血管病,尤其是脑出血,成为常见的危害人类身体健康的严重疾病[1]。该病具有发病急、进展快、致残率高、致死率高、并发症多等特点[2,3],截止目前为止,该病治疗方法较为局限,后期并发症发生率依然较高[4]。但通过科研人员在对脑出血发病机制的研究中也取得了一定的进展。促红细胞生成素(erythropoietin,EPO)是由肝脏和肾脏分泌的一种激素样物质,用于调节血细胞生成的一类细胞因子[5]。重组人促红细胞生成素(rhEPO)已规范生产。EPO的神经保护作用使其成为一个研究热点,其神经保护作用开始被广泛研究[6,7]。EPO治疗脑血管病的重要机制之一是保护神经元,改善神经元损伤。实验研究报道,EPO能够提高脑缺血患者的神经功能[8],但是EPO在脑出血中的研究较少,而且其具体作用机制不是很清楚,本课题探讨重组人促红细胞生成素rhEPO是否具有脑出血后神经系统保护作用及其相应机制。方法:将8周龄的Wistar雄性大鼠30只随机分3组(每组10只),组别为假手术组、脑出血ICH组、rhEPO治疗组,脑出血组大鼠进行ICH造模,假手术组除不注血外,其余步骤同ICH组,rhEPO组术后5分钟给予腹腔注射rhEPO,所有动物24小时后进行神经功能学评分,收集大鼠脑组织,利用原位缺口末端标记法检测神经细胞凋亡的变化;采用免疫组织化学SP法检测凋亡蛋白Caspase3表达;并采用Real-time PCR和Western blot检测磷酸化JAK2、磷酸化STAT5基因和蛋白表达情况。结果:手术建立脑出血模型后24小时进行神经功能评分。按Longa5分制标准判定,ICH组4只评价为1分,3只评价为2分,2只评价为3分;rhEPO组治疗后,5只评价为1分,2只评价为2分,1只评价为3分。说明rhEPO可以改善大鼠脑出血后神经元损伤,恢复神经功能。与假手术组相比,ICH模型组和rhEPO治疗组中神经元凋亡细胞及Caspase3的蛋白表达阳性细胞数明显增多(P0.05),而rhEPO组中凋亡细胞与ICH模型组相比明显减少(P0.05);与假手术组相比,ICH模型组和rhEPO组中JAK2和STAT5的蛋白表达和m RNA表达显著上升(P0.05),与ICH模型组相比,rhEPO组中JAK2和STAT5的蛋白表达和m RNA表达显著降低,差异均有统计学意义(P0.05)。结论:外源性rhEPO可以对脑出血后神经细胞起到保护作用,其作用机制是通过调节凋亡相关蛋白caspase以及JAK2/STAT5信号改善大鼠脑出血后神经元损伤。
[Abstract]:Background: with the acceleration of population aging, cerebrovascular disease, especially intracerebral hemorrhage, has become a common serious disease endangering human health [1].The disease is characterized by rapid onset, rapid progress, high disability rate, high fatality rate and many complications. Up to now, the treatment of the disease is limited, and the incidence of late complications is still high [4].However, some progress has been made in the study of the pathogenesis of intracerebral hemorrhage by researchers.Erythropoietin erythropoietin (EPO) is a hormone like substance secreted by the liver and kidney, which is used to regulate the production of blood cells [5].Recombinant human erythropoietin rhEPO (rhEPO) has made the neuroprotective effect of .EPO a hot research topic, and its neuroprotective effect has been widely studied. One of the important mechanisms for the treatment of cerebrovascular diseases is the protection of neurons, which is one of the important mechanisms for the treatment of cerebrovascular diseases by recombinant human erythropoietin (rhEPO).Improve neuronal injury.It is reported that EPO can improve the neurological function of cerebral ischemia patients [8], but there are few studies on the role of EPO in cerebral hemorrhage, and its mechanism is not very clear.The purpose of this study was to investigate whether recombinant human erythropoietin (rhEPO) has neuroprotective effect after intracerebral hemorrhage (ICH) and its mechanism.Methods: thirty 8-week-old male Wistar rats were randomly divided into 3 groups (10 rats in each group, sham-operated group, ICH group, intracerebral hemorrhage group, intracerebral hemorrhage group). The rats in the sham-operation group were treated with ICH, and no blood was injected into the sham-operation group.The other steps were the same as that in the ICH group. All the animals were given intraperitoneal injection of rhEPO 5 minutes after operation. After 24 hours, the neurological function scores were collected and the changes of neuronal apoptosis were detected by in situ Nick end labeling method.Immunohistochemical SP method was used to detect the expression of apoptotic protein Caspase3, and Real-time PCR and Western blot were used to detect the expression of phosphorylated JAK2, phosphorylated STAT5 gene and protein.Results: the neurological function was evaluated 24 hours after the establishment of ICH model.According to the standard of Longa5 score, 4 patients in ICH group were evaluated as 1 minute, 3 as 2 minutes, 2 as 3 minutes, 2 as 1 minute, 2 as 2 minutes and 1 as 3 points after treatment, 5 cases were evaluated as 1 minute, 2 cases were evaluated as 2 minutes and 1 case was evaluated as 3 points.The results showed that rhEPO could improve the neuronal injury and restore the neural function after intracerebral hemorrhage in rats.Compared with sham operation group and rhEPO group, the number of neuronal apoptotic cells and the expression of Caspase3 protein were significantly increased in rhEPO group, while the number of apoptotic cells in rhEPO group was significantly lower than that in ICH model group, and that in sham operation group was significantly lower than that in sham operation group, while the expression of Caspase3 protein in rhEPO group was significantly lower than that in ICH model group, while that in rhEPO group was significantly lower than that in ICH model group.The protein expression of JAK2 and STAT5 and the expression of m RNA in group A and rhEPO were significantly increased (P 0.05), and the protein expression of JAK2 and STAT5 and the expression of m RNA in group B were significantly lower than those in group B (ICH model group).The difference was statistically significant (P 0.05).Conclusion: exogenous rhEPO can protect neurons from intracerebral hemorrhage by regulating apoptosis-related protein caspase and JAK2/STAT5 signal to improve neuronal injury after intracerebral hemorrhage.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.34

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