肾源性细胞因子促红细胞生成素对急性脊髓损伤大鼠细胞凋亡的影响

Protective effect of erythropoietin on neurocyte apoptosis following experimental acute spinal cord injury in rats

目的:促红细胞生成素为肾源性细胞因子,在大鼠急性脊髓损伤后对脊髓神经功能具有保护作用。实验拟证明不同时间硬膜外腔注射后其对脊髓神经细胞凋亡的影响。方法:实验于2007-01/04在苏州大学附属第二医院动物实验室完成。①实验材料:清洁级成年雄性SD大鼠48只,体质量(270±10)g;实验用人重组促红细胞生成素为日本麒麟啤酒株式会社制造,规格3000IU/支。②实验分组及处理:将大鼠随机分为正常组6只,假手术组6只(仅切除椎板),生理盐水组18只,实验组18只。按改良Allen打击法建立大鼠脊髓不完全损伤模型。实验组分别于大鼠脊髓损伤后1,6,24h(每个时间点6只),于硬膜外腔注射重组人促红细胞生成素5000u/(kg·bw);生理盐水组于相同时间予以相同体积生理盐水。③实验评估:通过动物神经运动功能BBB评分及斜板试验评价神经损伤程度;苏木精-伊红染色法观察组织学改变,并采用原位末端标记法标记法检测脊髓神经细胞凋亡数。结果:①神经行为学评分:正常组、假手术组大鼠双下肢运动功能正常;与生理盐水组相比,脊髓损伤后实验组1,6及24h神经运动功能有改善;斜板角度及BBB评分均明显提高,差别有显著性意义(P<0.05)。②组织学苏木精-伊红染色结果:实验组组织学破坏改变明显较生理盐水组轻。③凋亡神经细胞及计数结果:实验组脊髓神经细胞凋亡指数均明显下降,差异有显著性(P<0.05)。结论:早期应用外源性促红细胞生成素对脊髓不完全损伤后引起的神经运动功能损害具有保护作用,能明显改善脊髓损伤后的临床功能表现。此种保护作用与促红细胞生成素能够抑制神经细胞凋亡有关。

AIM： Erythropoietin, a cell factor produced by kidney, can protect spinal nerve from acute spine cord injury （SCI） in rat. The experiment explored the effects of erythropoietin on the apoptosis of neural cells. METHODS： The experiment was performed in the animal laboratory of Second Affiliated Hospital of Soochow University from January to April 2007. ①Forty-eight adult male Sprague-Dawley rats of （270±10） g were randomly divided into normal group （n =6）, sham operation group （n =6） underwent laminectomy procedure only, saline group （n =18） and treatment groups （n =18）. The SCI models were established using the improved Allen method. The rats in treatment group were epidurally injected with 5 000 u/kg body mass of recombinant human erythropoietin （Kirin Beer Company, Japan, 3 000 IU per piece） 1, 6, and 24 hours after injury respectively （6 animals at each time point）. Control group received normal saline epidurally at the same time point. ③Behavioral evaluation of the rats was made 48 hours after trauma by using the Basso, Beattie, and Bresnahan （BBB） scoring system and Rivlin＇s tiltboard experiment; Hematoxylin and eosin （HE） staining was performed for histological observation after injury. Injured spinal cord tissues cells apoptosis were examined by the terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling （TUNEL） reaction. RESULTS： ①Neuroethology evaluation： The both lower extremities of rats in normal group and sham operation group showed normal movement function. Compared to saline group, neuromotor function in rats after SCI was significantly improved in 1, 6 and 24 hours, and the angles of Rivlin＇s tiltboard experiment and the scores of BBB were significantly increased （P 〈 0.05）. ②HE staining results suggested that the histological damage extent of the spine cord in treatment group was remarkably lessened compared with saline group. ③ TUNEL staining： The number of TUNEL-positive cells was significantly decreased in the erythropoietin-treated rats （P 〈 0.05）. CONCLUSION： Early application of recombinant human erythropoietin can lessen the lesion of neuromotor function, and significantly improve the clinical function after SCI. The protection is partially due to the reduction of neural cell apoptosis.