冷冻预处理温度对胎兔许旺细胞活性的影响

Effect of frozen pretreatment temperature on bioactivity of embryonic rabbit's Schwann cells

背景:许旺细胞作为周围神经损伤修复的重要桥接材料,其活性对临床修复周围神经损伤成功与否具有重要影响。目的:观察不同维持温度冷冻预处理超深低温保存对胎兔许旺细胞活性的影响。方法:设计胎兔许旺细胞冷冻维持温度为-30～-70℃,每间隔-5℃为一组,以10%二甲基亚砜为低温保护剂,对胎兔许旺细胞进行上述温度冷冻预处理,液氮中保存48h,快速复温后继续培养48h,透射电镜观察细胞超微结构改变,MTT法和3H-TdR掺入法检测细胞活性。结果与结论:透射电镜下见-45℃处理的细胞超微结构良好,其余各温度组细胞均出现细胞器肿胀、坏死等轻重不同的冷冻损伤表现。MTT法和3H-TdR检测结果均显示-45℃冷冻预处理对许旺细胞的生长抑制率最低,分别为(3.83±0.56)%、(4.41±0.71)%,和其余各温度组之间相比,差异有显著性意义(P<0.05)。提示-45℃是胎兔许旺细胞冷冻预处理的最佳维持温度值,对细胞活性影响最小,可较好保持其生物学活性。

BACKGROUND：Schwann cells are considered as an important bridge material for peripheral nerve repair,and the activity of Schwann cells is a key to whether the repair of peripheral nerve injury succeeds or not.OBJECTIVE：To investigate the effect of different frozen pretreatment temperatures on the bioactivity of embryonic rabbit＇s Schwann cells preserved at ultra-low temperature.METHODS：The embryonic rabbit＇s Schwann cells were frozen at-30 ℃,-35 ℃,-40 ℃,-45 ℃,-50 ℃,-55 ℃,-60 ℃,-65 ℃ as well as-70℃ respectively,treated by a cryoprotectant,10% dimethyl sulfoxide,at the same time,preserved in liquid nitrogen for 48 hours,and cultured for 48 hours after rapid rewarming.Morphological changes of Schwann cells were observed under transmission electron microscopy（TEM）.Bioactivity of Schwann cells was assessed by MTT and 3H-TdR incorporation test assays.RESULTS AND CONCLUSION：The ultrastructure of Schwann cells in the group of-45 ℃ remained intact under TEM;however,other experimental cells exhibited the different appearances of cryodamage such as swelling organelles,necrosis in the cytoplasm.Results in MTT and 3H-TdR assays showed that growth inhibiting rate in the group of-45 ℃ was（3.83±0.56）% and（4.41±0.71）%,which was the lowest among the experimental groups（P〈0.05）.-45 ℃ is the optimal frozen pretreatment temperature that can protect the bioactivity of embryonic rabbit＇s Schwann cells.