深低温保存兔肢体不同方法复温后许旺细胞的活性

摘要

BACKGROUND:According to the characteristics of nerve regeneration, the integrity of Schwann cels as regenerative scaffold is very important. Therefore, to save the Schwann cel structure and activity is most important for nerve cryopreservation. OBJECTIVE: To investigate the optimal thawing procedures for the survival of peripheral nerves in cryopreserved rabbit limbs. METHODS:Forty hindlimbs from New Zealand White rabbits were randomized into four groups: blank, fast-thawing, slow-thawing and control groups. The control group was subjected to perfusion with protective agent. The severed limbs in fast-thawing and slow-thawing groups were perfused with protective agent folowed by gradient cryopreservation and storage in liquid nitrogen for 72 hours. Then, the slow-thawing group was given gradient thawing procedures, and the fast-thawing group was subjected to fast thawing in 37℃ water bath. After thawing, the tibial nerves were harvested for light microscope, electron microscope, immunofluorescence and confocal microscope observation. SPSS17.0 was used for statistical analysis. RESULTS AND CONCLUSION:No significant difference was found between the blank and control groups. Nerve fiber breakage, axonal damage and mitochondrial sweling were severer in the fast-thawing and slow-thawing groups than the control and blank groups. In addition, the fast-thawing group was superior to the slow-thawing group. These findings indicate that the fast-thawing method has the minimal effects on the survival of nerve fibers in cryopreserved rabbit limbs.%背景:根据神经再生的特点,作为再生支架的许旺细胞完整性十分重要,因此神经的深低温保存最重要的是保存许旺细胞的结构和活性.目的:使用不同方法复温深低温保存的兔肢体,通过对比其神经组织的微观形态学变化,得到一种对深低温保存复合组织中的神经组织损伤较小的复温方法.方法:取40个新西兰大白兔后肢,随机分为空白组、快速复温组、慢速复温组、对照组.空白组不进行任何处理,对照组给予保护剂灌注,快速复温组、慢速复温组给予保护剂灌注后采用梯度化降温处理并保存于液氮中72 h,而后慢速复温组采用梯度化复温断肢,快速复温组将断肢直接放入37℃水浴中快速复温.复温后的各组肢体切取胫神经,采用光镜、电镜、免疫荧光、共聚焦显微镜进行观察对比,结果用SPSS 17.0统计软件进行统计分析.结果与结论:通过光镜、电镜、免疫荧光、共聚焦显微镜观察对比,空白组与对照组比较差异无显著性意义,快速复温组、慢速复温组两组均有不同程度的神经纤维断裂,轴突破坏以及线粒体肿胀,与空白组、对照组两组比较差异有显著性意义,快速复温组与慢速复温组比较,快速复温组显著优于慢速复温组.结果说明,深低温保存的兔断肢,快速复温方法对复合组织中的神经组织损伤较小.