【作者】 刘君；

【导师】 周立军； 江荣才；

【作者基本信息】 天津大学 ， 微生物与生化药学， 2010， 硕士

【摘要】 目的:研究重组人源红细胞生成素诱导创伤性脑损伤Wistar大鼠模型循环血内皮祖细胞动员、促进创伤后脑组织神经血管修复的作用及其潜在的细胞学机制。方法:健康雄性Wistar大鼠174只,体重300-350g,随机分为I批(n=144)和II批(n=30)。I批分为Sham(假损伤)组、TBI~+Saline(生理盐水)组及TBI~+rhEPO(重组人源红细胞生成素)组,每组均n=48。I批三个组再根据TBI动物模型的制模时间,设定伤后3h,6h,24h,3d,7d,14d共6个观测时间点,每个时间点均随机选取大鼠n=8只。II批分为Sham组、TBI~+Saline组及TBI~+rhEPO组,每组均有大鼠n=10。大鼠TBI模型制备采用液压颅脑损伤仪,给予1.5-2.0atm液压冲击力,制成中型液压颅脑损伤模型。分别于致伤后相应观察时间点对I批各组大鼠进行眼内骴静脉取血、断头取脑。采用流式细胞仪检测循环血中CD34~+、CD133~+细胞即EPCs的水平,采用免疫荧光染色观察TBI创伤组织海马区和皮质区的CD31~+细胞的表达变化,即新生血管内皮细胞的变化。同时,对II批各组大鼠进行Morris水迷宫试验和神经功能损伤评分实验,以分别评定TBI大鼠在不同干预条件下空间学习和记忆能力变化和rhEPO对TBI大鼠神经功能修复作用。结果:1.与Sham组相比,TBI~+Saline组及TBI~+rhEPO组大鼠循环血EPCs水平总体上呈现先低后高的变化趋势,但TBI~+rhEPO组6h后循环血EPCs明显较TBI~+Saline组升高,且维持高EPCs水平至7d达到最高点,然后开始降低,相反,TBI~+Saline组循环血EPCs的水平损伤后即呈低水平,然后开始逐渐升高,到6h后开始下降,直至正常水平。2.与Sham组相比,伤后1d时间点的TBI~+Saline和TBI~+rhEPO组海马区和皮质区的CD31~+表达明显增加,TBI~+Saline和TBI~+rhEPO组相比无统计学意义。伤后7d时间点的TBI~+Saline和TBI~+rhEPO组海马区和皮质区CD31~+表达也比sham组相比增强,与TBI~+Saline相比,TBI~+rhEPO组大鼠CD31~+表达增高且具有统计学意义。3.水迷宫实验显示,整体上各组大鼠随着训练天数的增加,在目标象限所花时间的百分率逐渐增加。与TBI~+Saline组相比,TBI~+rhEPO老鼠的目标象限百分率从第四天明显增加(P<0.05)。神经功能损伤评分实验显示TBI~+rhEPO从第14天开始其NSS评分与TBI~+Saline组相比显著降低(P<0.05),第21天与对照组相比,神经功能改善更为明显(P<0.01),说明rhEPO能增强创伤性TBI大鼠的血管生成和空间学习记忆能力。结论:rhEPO可以上调创伤性脑损伤大鼠循环血EPCs的水平,并促进创伤组织的血管修复,增强TBI大鼠的空间学习记忆能力,可能是脑创伤的潜在治疗药物。更多还原

【Abstract】 Objective: To explore the inducing effect of the recombinant human erythropoietin (rhEPO) on the level of circulating endothelial progenitor cell (EPCs) in the Wistar rat models of traumatic brain injury(TBI), on the neuro-vascular repairing effect and on the potential cellular mechanisms.Methods: 174 health male Wistar rats (8-week-old, weight: 300g-350g) were divided randomly into group I (n=144) and group II (n=30). Group I was further divided into three subgroups: Sham injury, TBI~+Saline and TBI~+rhEPO respectively (n=48 for every group). To assay the level of the circulating EPCs, 6 investigating time points were set as 3h, 6h, 24h, 3d, 7d, 14d post-trauma and each point contains 8 rats. Gorup II was also divided into tree subgroups: Sham injury, TBI~+Saline and TBI~+rhEPO respectively (n=10 for every group). The TBI rat models were made by attacking the opened cranial of the rats with a fluid-percussion power (1.5-2.0atm) of the fluid-percussion craniocerebral injury instrument. Blood samples of the rats were collected from their posterior orbits and the brain tissues were collected at each corresponding time point after trauma. Marked with both positive CD34 and CD133 antibodies, the level of the circulating EPCs was assayed in the flow cytometry instrument. The immunofluorescence staining was also performed to investigate the expression changes of CD31~+ on hippocampus and cortex of the injured brain tissue, which is the marker of the endothelial cells of the matured vascular indicating a degree of the vascular repairing. The spatial learning and memory ability of the rats from the subgroups of group II was assessed to assay the rhEPO therapeutic effect on the neuro-function recovery of the TBI rats.Results: 1. Compared with sham group, in general, the level of circulating EPCs in TBI~+Saline and TBI~+rhEPO Wistar rats post-TBI decreased immediately, then increased later. The level of EPCs of TBI~+rhEPO rats were significantly higher than the one of the TBI~+Saline 6h post-TBI, the former retained high until 7d and then decreased to normal level several days later. However, the level of the EPCs of TBI~+Saline rats peaked at 6h after trauma and decreased to a normal level thereafter. 2. The immunofluorescence staining of CD31~+ in cortex and hippocampus tissues was stronger in TBI~+Saline and TBI~+rhEPO rats than in sham rats 1day post-TBI, however, no significant difference was detected between the TBI+Saline and TBI+rhEPO rats. Another 6 days later(7 d post-trauma), the CD31 expression in brain tissue from both TBI+Saline and TBI+rhEPO rats was more significant than the sham rats, with the highest one in the TBI+rhEPO rats when compared with the TBI+Saline rats (P<0.05). 3. In general, the percent time spending in target quadrant increasesd following the days practiced in the Morris water maze test. Compared with the TBI+Saline rats, the percent time spending in the target quadrant of the TBI+rhEPO rats increased apparently from the fourth days on (P<0.05), which indicates that the rats treating with rhEPO improved their ability of spatial learning and memory. In addition, the total neurological severity scores of the TBI+rhEPO rats were lower than TBI+Saline rats, even much lower on 21d post-trauma, which also indicates that the rats treating with rhEPO improved their severity of TBI rats.Conclusion: rhEPO could increase the number of circulating EPCs, promote the neurovascular repair in injured brain tissue and improve spatial learning ability and memory functionh of the TBI rats. It may be a potential therapeutic drug to cure TBI.更多还原