神经干细胞单细胞与神经球治疗大鼠脊髓损伤的对比研究  

COMPARATIVE STUDY ON SINGLE CELL SUSPENSION OF NEURAL STEM CELLS AND NEUROSPHERES TRANSPLANTATION FOR SPINAL CORD INJURY IN RATS

作　　者：钟林[1] 张辉[1] 高维陆[1] 尹宗生[1] 

机构地区：[1]安徽医科大学第一附属医院骨科,合肥230022

出　　处：《中国修复重建外科杂志》2012年第7期855-860,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30973026);安徽省自然科学基金资助项目(11040606025)~~

摘　　要：目的比较神经干细胞(neural stem cells,NSCs)单细胞与神经球移植治疗大鼠脊髓损伤(spinal cordinjury,SCI)的效果,研究两种NSCs移植方法治疗SCI的有效性。方法取成年SD大鼠2只,体外分离脊髓组织并行NSCs培养,取第3代细胞行Hoechst33342、巢蛋白染色及贴壁分化鉴定。另取成年SD大鼠(体重230～250 g)60只,随机分为3组,每组20只,采用改良Allen法制备大鼠脊髓T10损伤模型。各组分别于SCI处缓慢注射5μL生理盐水(A组)、第3代NSCs单细胞(B组)、第3代NSCs神经球(C组)。分别于术前及术后3、7、14、21、28 d采用BBB行为功能评定量表评定各组大鼠后肢功能;术后各时间点取材行HE染色和微管相关蛋白2(microtubule-associated protein 2,MAP-2)免疫组织荧光染色观察。结果经形态学观察及鉴定,所培养的细胞为NSCs。术后3 d各组BBB评分较术前显著下降,术后3、7 d各组间BBB评分比较差异均无统计学意义(P>0.05);随时间延长BBB评分不同程度增加,术后14、21、28 d,B、C组BBB评分优于A组,C组优于B组,比较差异均有统计学意义(P<0.05)。HE染色示C组较A、B组脊髓结构清晰,瘢痕形成少。MAP-2免疫组织荧光染色示,术后3、7 d各组间阳性细胞数比较差异均无统计学意义(P>0.05);术后14、21、28 d,B、C组阳性细胞数显著多于A组,C组多于B组,差异有统计学意义(P<0.05)。结论采用NSCs神经球移植较单细胞移植更明显促进NSCs向神经元分化,更有利于SCI后下肢功能恢复。Objective To compare single cell suspension of neural stem cells （NSCs） with neurospheres transplantation for spinal cord injury （SCI） so as to explore the therapeutic effectiveness of two NSCs transplantation methods for SCI. Methods The NSCs were isolated from the spinal cord of adult Sprague Dawley （SD） rats, purified and cultured. At passage 3, the cells were identified by Hoechst33342, Nestin staining, and glial fibrillary acidic protein staining for differentiated cells. Sixty adult SD rats （weighing 230-250 g） were made the SCI models at T10 level with modified Allen method and randomly divided into 3 groups （20 rats in each）. The injury sites were treated by injecting 5 μL saline （group A）, 5 μL single cells suspensions of NSCs at passage 3 （group B）, and 5 μL neurospheres cell suspensions at passage 3 （group C）. At preoperation and 3, 7, 14, 21, and 28 days after operation, the locomotor functions of each group were assessed using the Basso, Beattie, and Bresnahan （BBB） rating scale. HE staining was applied to observe the morphology of spinal cord. Subsequently immunofluorescence staining was used to observe microtubule-associated protein 2 （MAP-2）. Results The cells cultured were NSCs by morphological observation and immunofiuorescence staining. After 3 days of modeling surgery, BBB score significantly decreased when compared with preoperative score, and there was no significant difference among 3 groups at 3 and 7 days （P 〉 0.05）. BBB score increased in different degrees with time; at 14, 21, and 28 days, BBB score of groups B and C was better than that of group A, and group C was better than group B, showing significant differences （P 〈 0.05）. HE staining showed that spinal cord structure of group C was more clear than that of groups A and B, and had less scar. There was no significant difference in the number of MAP-2 positive cells among 3 groups at 3 and 7 days （P 〉 0.05）. At 14, 21, and 28 days, the number of MAP-2 positive cells of grou

关 键 词：神经干细胞 脊髓损伤 细胞移植 神经球 大鼠 

分 类 号：R651.2[医药卫生—外科学]