【摘要】 目的:观察微囊化兔嗅球组织细胞移植对大鼠脊髓全横断损伤后细胞凋亡及功能恢复的影响,并探讨其对脊髓继发性损伤的保护作用及其机制。方法:实验于2004-10/2005-07在南昌大学基础医学院应用解剖研究室完成。选择清洁级SD大白鼠120只,按随机数字表法分为4组:正常对照组、损伤对照组、单纯细胞悬液移植组、微囊化移植组,每组30只。制作T10节段脊髓全横断损伤模型。正常对照组仅打开椎管,暴露脊髓不作移植;损伤对照组仅用明胶海绵填塞脊髓损伤腔隙;单纯细胞悬液移植组植入吸附细胞悬液的明胶海绵块;微囊化移植组用与断端腔隙大小相吻合的明胶海绵块吸附10μL的微囊化细胞,植入洞腔。分别于术后12h,1,3,7,21d5个时间点对动物进行BBB评分后处死。取损伤区1cm范围脊髓节段,常规石蜡包埋,水平切片进行苏木精-伊红染色、TUNEL染色,观察凋亡细胞的数量及分布变化。按BBB评分法对大鼠术后运动功能的恢复情况进行评分,共分22级,最低分0分,最高分21分,分数越高,运动功能恢复越完善。结果:纳入大鼠120只,存活96只,存活率为80%。其中以损伤对照组及单纯细胞悬液移植组死亡率较高,损伤对照组大鼠死亡的主要原因可能是脊髓损伤导致损伤平面以下神经功能的丧失所引起的一系列并发症所导致的;而单纯细胞悬液移植组是因为免疫排斥反应所导致。①术后3d内各组间BBB评分差异无显著性意义(P>0.05);术后7,21d微囊化移植组和单纯细胞悬液移植组大鼠BBB评分高于损伤对照组,差异均有显著性意义[分别为(7.25±1.17),(4.58±0.38),(2.67±0.61)分;(10.42±1.63),(6.08±0.80),(3.33±0.68)分,F=4.528,3.821,P<0.05],且经微囊化处理后比单纯兔嗅球组织细胞悬液移植运动功能恢复更好(F=4.112,P<0.05);而损伤对照组大鼠后肢运动功能恢复均较差。②光镜下损伤对照组、单纯细胞悬液移植组脊髓损伤后,脊髓内空洞增大,损伤范围基本确定,周围有炎性细胞浸润;在微囊化移植组脊髓损伤明显减轻,细胞皱缩不明显、胞质、胞核较清晰。③术后1,3,7d微囊化移植组TUNEL阳性细胞数及凋亡指数低于损伤对照组,差异有显著性意义(以术后3d为例,TUNEL阳性细胞数分别为(8.83±1.33),(14.50±1.05)个/视野,P<0.01;凋亡指数分别为10.45±1.58,17.06±1.23,P<0.01)。结论:微囊化兔嗅球组织细胞移植对大鼠脊髓全横断损伤后细胞凋亡具有保护作用,能促进脊髓功能的恢复。更多还原

【Abstract】 AIM:To observe the effect of microencapsulated rabbit olfactory bulb xenotransplantation on cell apoptosis and functional recovery after transected spinal cord injury(SCI) in rats,and explore its protective mechanism during the period of secondary affection.METHODS:The experiment was accomplished in the Department of Human Anatomy,School of Basic Medical Science,Nanchang University between October 2004 and July 2005.A total of 120 SD clearing rats were randomly divided into 4 groups:normal control group,injury control group,cell grafted group,microcapsuled cell grafted group,with 30 rats in each.The experimental injury models of SD rats were made by transected SCI at the T10 level.Normal control group:Vertebral canal was opened to expose spinal cord without transplantation;Injury control group:Transected spinal lacouna was packed with gelatin sponge;Cell grafted group:Transplantation was conducted with gelatin sponge loaded with cell suspension;Microcapsuled cell grafted group:Spinal cavity was packed by gelatin sponge which was identical with broken spinal cavity in dimension and loaded 10 μL microencapsulated cells.Rats were sacrificed at 12 hours and 1,3,7,21 days after injury,a 1-cm cord segment centered at the lesion site was removed and embedded in paraffin.Serial 5 μm cross-section were cut and stained with the methods of TUNEL and hematoxylin-eosin(HE) before each rat was sacrificed,in order to observe the number and distribution of apoptosis cells.BBB scale was designed to assess the behavioral ability of rats after operation in 22 grades ranged from 0 to 21,and the higher scores indicated the better functional recovery.RESULTS:All 120 rats were involved in the experiment,and 96 of them survived after transplantation,accounting for 80%.The mortality was higher in both injury control group and cell grafted group,due to a series of complications induced by neurofunctional loss after SCI or immunological rejection.①There was no significant difference in BBB scores among four groups before 3-day postoperation(P > 0.05) ;BBB scores were higher in microcapsuled cell grafted group and cell grafted group than in injury control group [(7.25±1.17) ,(4.58±0.38) ,(2.67±0.61) scores for 7-day;(10.42±1.63) ,(6.08±0.80) ,(3.33±0.68) scores for 21-day,F = 4.528,3.821,P < 0.05],and the motor functional recovery of rats was better in microcapsule grafted group than in cell grafted group(F = 4.112,P < 0.05) ,but not satisfied in injury control group.②In injury control group and cell grafted group,the spinal cavity increased and inflammatory cell infiltrated after SCI;However,SCI was relieved in microcapsuled cell grafted group,there were a littlecellular shrank,and the cytoplasm and nucleus presented clearly.③At 1,3,7 days postoperation,the TUNEL-positive cell number and apoptosis index were both significantly lower in microcapsuled cell grafted group than in injury control group [(8.83±1.33) ,(14.50±1.05) cells/field,P < 0.01;10.45±1.58,17.06±1.23,P < 0.01].CONCLUSION:Microcapsulated rabbit olfactory bulb xenotransplantation can inhibit the apoptosis after SCI and improve the functional recovery of spinal cord in SD rats.更多还原