低温保存神经干细胞复苏后移植对脊髓损伤大鼠轴突再生的影响:逆行示踪标记观察  

Effects of cryopreserved neural stem cell transplantation on rat axonal regeneration after spinal cord injury:Retrograde tracing observation

作　　者：杨永利[1] 刘宏志[1] 李佐文[1] 

机构地区：[1]铁法煤业集团总医院骨科,辽宁省调兵山市112700

出　　处：《中国组织工程研究与临床康复》2008年第3期447-450,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：教育部高校博士点专项科研基金项目(20060159019);辽宁省自然科学基金资助项目(20052096)~~

摘　　要：目的:关于神经干细胞移植治疗脊髓损伤的研究已有一些报道,对细胞移植的时间、方式以及检测的指标各有不同观点。实验观察了低温保存的神经干细胞复苏后移植对大鼠脊髓损伤后轴突再生的影响。方法:实验于2005-06/2006-06在中国医科大学实验动物中心完成。①实验材料:选取Wistar成年大鼠36只,雌雄不限,体质量250～300g,由中国医科大学实验动物部提供。新生大鼠10只,用作神经干细胞的分离与培养。实验过程中对动物处置符合动物伦理学标准。②实验方法:将获得的神经干细胞在处于对数生长期阶段-70℃冻存2周,复温后用5-溴脱氧尿嘧啶核苷法标记。制备大鼠脊髓损伤模型,伤后立即进行移植干预,实验分为3组:神经干细胞移植组、DMEM培养液填充组、空白对照组。③实验评估:应用免疫组织化学法观察移植细胞存活及迁移情况,行辣根过氧化物酶逆行示踪法观察脊髓损伤处轴浆运输的重建。结果:36只脊髓横断损伤模型因麻醉过量死亡4只,感染死亡5只,予补足。复苏的神经干细胞经Brdu核标记后移植到脊髓损伤区,在损伤脊髓区域可检测到标记的阳性细胞。第7天可见,第14天增多,第28天逐渐减少并消失。辣根示踪技术显示神经干细胞移植组较DMEM培养液填充组阳性细胞明显增多,组间差异有统计学意义。结论:低温保存的神经干细胞移植到脊髓损伤区域后可存活,并参与脊髓损伤处轴浆通路的结构重建。AIM： There are some reports about the treatment of spinal cord injury with neural stem cells （NSCs） transplantation, but the ideas about the transplantation time and patterns and detection indexes are still different. In this study, the effects of cryopreserved NSC transplantation on the axonal regeneration after the spinal cord injury in rats were observed. METHODS： The experiment was carded out in the experimental animal center of China Medical University from June 2005 to June 2006. （1）Thirty-six adult Wistar rats, either male or female and 250-300 g, were provided by the experimental animal department of China Medical University. Neural stem cells were isolated from 10 neonate rats and cultured. All treatments for animals were accorded with the animal ethical criteria. （2）NSCs at logarithmic phase were cryopreserved at -70 ℃ for 2 weeks and labeled with 5-bromodeoxyuridine （Brdu） after rewarming. After the models of spinal cord injury were established, the NSCs were transplanted into the injured site immediately. Thirty-six rats were randomly divided into NSC transplantation group, DMEM solution group, and control group. （3）NSC survival and migration were detected by immunohistochemistry, and the reconstruction of spinal cord were detected by horseradish peroxidase （HRP） staining. RESULTS： Of the 36 spinal injured model rats, 4 died of overdose of anesthesia, and 5 died of infection, but all were supplemented. Brdu-labeled positive NSCs were detected in the injured spinal cord after transplantation on the 7th day, and increased on the 14th day, then gradually decreased since the 28th day till disappeared, The number of HRP positive cells in transplantation group was significantly higher than DMEM solution. CONCLUSION： Cryopreserved NSCs can survive in the injured site after transplantation and promote the reconstruction of axoplasm pathway of injured spinal cord.

关 键 词：脊髓损伤 神经干细胞 标记 轴突再生 低温保存 

分 类 号：R651.2[医药卫生—外科学]