【作者】 李鑫；

【导师】 朱涛；

【作者基本信息】 天津医科大学 ， 外科学， 2011， 硕士

【摘要】 目的：建立大鼠脊髓半横断损伤模型,并于硬膜下腔给予重组人促红细胞生成素(rh-EPO),研究rh-EPO对大鼠脊髓损伤后运动功能恢复、病理改变及神经细胞凋亡的影响,探讨rh-EPO治疗脊髓损伤的治疗效果、最佳治疗剂量及二者之间的关系,为临床治疗急性脊髓损伤提供实验依据。方法：1.脊髓半横断损伤模型：在显微镜下,应用显微刀片对大鼠T10脊髓行右侧半横断损伤,并于硬膜下腔放置自制注射管。2.将80只成年雄性Wistar大鼠随机分为5组(n=16),即假手术组、NS对照组、小剂量EPO治疗组、中剂量EPO治疗组、大剂量EPO治疗组,除假手术组外其余均行脊髓半横断损伤。其中假手术组仅行椎板去除,不予药物治疗；NS对照组按照不同给药时间点(术后30min内、术后ld、术后3d、术后7d)于硬膜下腔置管内缓慢注射生理盐水,每次50μl；小、中、大剂量EPO治疗组按上述给药时间点于硬膜下腔置管内给予rh-EPO。每次给药时,分别将100IU/kg、500IU/kg、1000IU/kg rh-EPO加枸橼酸纳缓冲液至50μl后,相应缓慢地注入小、中、大剂量EPO治疗组大鼠硬膜下腔。各组大鼠均于术后14d处死。3.所有大鼠均于伤前、伤后定期进行BBB行为学评分,连续观察2w脊髓损伤大鼠后肢运动功能恢复情况。大鼠处死后,损伤脊髓组织经石蜡包埋切片,采用HE染色观察其病理改变,并应用原位缺口末端标记法(TUNEL法)标记损伤组织凋亡细胞,在光镜下统计并分析结果,计算凋亡指数(Apoptotic Index, AI)。采用t检验(Student’s t-test)比较两组数据之间的差异性,用方差分析(analysis of variance, ANOVA)比较多组数据间的差异性,p<0.05即认为差异有统计学意义,P<0.01即认为差异具有显著统计学意义。结果：1.大鼠脊髓半横断损伤模型成功稳定,重复性好，且伤口整齐,损伤程度恒定,解剖定位准确,功能障碍亦确定2.BBB评分结果显示：①与术后1d相比较,各治疗组术后3d、7d及14d的BBB评分明显增加(P<0.01)；②小剂量EPO治疗组较NS对照组BBB评分增加(P<0.05)；③中、大剂量EPO治疗组与NS对照组及小剂量EPO组相比较,BBB评分增加明显(P<0.01)；④中、大剂量EPO治疗组之间比较,BBB评分无明显改变(P>0.05)。3.HE染色显示：①假手术组大鼠脊髓组织结构正常,神经元形态完好,尼氏小体清晰可见,外周白质区轴突正常。②损伤组表现为脊髓组织水肿、弥漫性出血,囊腔及空泡形成,神经元变性乃至消失,尼氏小体分辨不清,周围大量炎症细胞浸润,星形胶质细胞大量增生,胶质瘫痕形成,轴索退变,轴索与髓鞘之间空隙加大。③EPO治疗组创伤病理表现较NS对照组轻；中、大剂量EPO组较小剂量组轻。4.凋亡细胞检测结果显示：假手术组偶见阳性细胞；损伤组白质中及空洞周边可见大量阳性细胞表达。与NS对照组相比较,小剂量EPO组的凋亡细胞数及AI降低(P<0.05)；中、大剂量EPO组明显低于NS对照组及小剂量EPO组(P<0.01)；中、大剂量EPO组之间比较,凋亡细胞数及AI无明显差异(P>0.05)。5.相关性分析显示：BBB评分有随凋亡细胞数目减少而增加的趋势,二者呈负相关。结论：1.大鼠脊髓半横断损伤模型适于进行脊髓损伤的基础理论研究和神经生物学观察。2. rh-EPO有助于脊髓损伤大鼠BBB运动功能评分的增加,促进大鼠运动功能恢复。3. rh-EPO能改善大鼠脊髓损伤组织的病理改变。4. rh-EPO能减轻大鼠脊髓损伤后神经细胞凋亡,降低凋亡指数。5.BBB评分有随凋亡细胞数目减少而增加的趋势。6.中、大剂量rh-EPO治疗脊髓损伤,效果优于小剂量rh-EPO;中等剂量rh-EPO适于治疗脊髓损伤。更多还原

【Abstract】 Objective:To establish the Wistar rats spinal cord hemisection injury model, the damage region was applied recombinant human erythropoietin (rh-EPO) through subdural injection. Then to study the effection of rh-EPO on motor function recovery, pathological changes and neuronal apoptosis after spinal cord injury (SCI), and to discuss optimal treatment dose, the therapeutic effect and the dose-response relationship about EPO treatment of SCI, so as to provide experimental evidence for the clinical treatment of SCI.Methods:1. Spinal cord hemisection injury model:under the microscope, the right-half side of T10 spinal cord in rats was cut with microscopic blade and self-made injection tube was placed to the subdural.2.80 adult male Wistar rats were randomly divided into 5 groups (n=16):sham group, NS control group, low-dose EPO treatment group, middle-dose EPO treatment group and high-dose EPO treatment group. All groups underwent spinal cord hemisection except sham group. Sham operation group in which the line lamina removed, was no drug treatment; NS control group, according to different administration time points (within 30min after surgery, Id after surgery,3d after surgery and 7d post-operative), was injected slowly with saline 50μl to the subdural catheter every time; Small, medium and high doses EPO treatment group were given rh-EPO to the subdural catheter in accordance with the above delivery time points. Each time when the drug was given, 100IU/kg,500IU/kg, 1000IU/kg rh-EPO were respectively added sodium citrate buffer solution to 50μl, and then were injected slowly into the rats subdural of small, medium and high doses EPO treatment group accordingly. Rats in all groups were sacrificed after 14d.3. All the rats’behavial changes were valued regularly with the BBB scale before and after injury, and the motor functional recovery of rat hindlimb after SCI was observed continuously for 2 weeks. After sacrificed, the injuried spinal cord tissues of rats were paraffin-embedded and cutted into slices. Then the pathological changes were observed with hematoxylin-eosin staining, the apoptotic cells of the injuried spinal cord tissue were labelled with TUNEL method, the results were analyzed under the light microscope and the apoptotic index was calculated. Data analysis was performed with Student’s t-test and one-way ANOVA, P<0.05 is considered statistically different, and P<0.01 is considered statistically significant different.Results:1. Hemisection model of spinal cord is successful, stable and repeatable. The wound is neat, degree of injury is constant, anatomical localization is accurate, and functional disorders are also identified.2. BBB score showed:①Compared with Id after surgery, BBB scores in all treatment groups increased significantly on 3d,7d and 14d after surgery (P< 0.05).②BBB scores of low-dose EPO treatment group were higher than the NS control group (P<0.05).③Medium and high dose EPO treatment group compared with the NS control group and low-dose EPO group, BBB scores increased significantly (P<0.01).④Comparing medium and high dose EPO treatment groups, BBB scores had no significant change (P>0.05).3. HE staining showed:①Sham group:Rat spinal cord tissue was normal, neuronal morphology were intact, nissl bodies were clearly visible, and axons of peripheral white matter was normal.②Injuried groups:There were spinal cord edema, diffuse hemorrhage, cysts and vacuoles formation, neuronal degeneration and even disappearance, confusion Nissl bodies, large proliferation of astrocytes, glial scar formation, axonal degeneration appeared. Neurons were surrounded by a large number of inflammatory cells and the gap between axon and myelin was larger.③The pathological damage of EPO treatment group was milder than the NS control group, and the pathological damage of medium and high dose group was lighter than smaller-dose EPO.4. Apoptotic cells results showed:Positive cells were seen occasional in the sham group, and there was a large number of positive cells in the white matter and around the lesion in injuried groups. Compared with the NS control group, the number of apoptotic cells and AI of low-dose EPO group were decreased (P< 0.05). Medium and high dose EPO treatment group compared with the NS control group and low-dose EPO group, the number of apoptotic cells and AI decreased significantly (P<0.01). Comparing medium and high dose EPO treatment groups, the number of apoptotic cells and AI had no significant difference (P>0.05).5. Correlation analysis showed:BBB score increased with the decreased number of apoptotic cells, and there was a negative correlation between the two.Conclusion:1. Rats spinal cord hemisection model is suitable to conduct fundamental research and neurobiology observation of SCI.2. rh-EPO helps rats after SCI with increasing BBB motor functional scores, and promotes recovery of motor function in rats.3. rh-EPO can improve the pathological changes of spinal cord injuried tissue in rats.4. rh-EPO can reduce neuronal apoptosis after spinal cord injury in rats, and reduce the apoptotic index.5. There was a trend that BBB score increased with the decreased number of apoptotic cells.6. Medium, high-dose rh-EPO treatment of spinal cord injury is better than low-dose rh-EPO; Medium-dose rh-EPO is optimal treatment dose of spinal cord injury.更多还原