摘要： 目的·探讨热休克蛋白70（heat shock protein 70，HSP70）对冷冻保存大鼠坐骨神经细胞活性及异体移植后神经再生的影响。方法·将SD大鼠双侧坐骨神经置于DMEM培养基中，分别于37、42、45 ℃体外预处理1 h（记为37 ℃组、42 ℃组、45 ℃组），并设置未经热应激处理的对照组（Con组），每组神经36根。蛋白质印迹（Western blotting）检测各组坐骨神经中HSP70的表达。将上述4组神经于液氮冷冻保存液中保存4周，Western blotting检测主要组织相容性复合体Ⅰ（major histocompatibility complex class Ⅰ，MHC-Ⅰ）、MHC-Ⅱ和半胱氨酸天冬氨酸蛋白酶3（caspase-3）、caspase-9的表达，流式细胞术检测坐骨神经活细胞存活情况。取冷冻保存的坐骨神经，于37 ℃、5% CO
2培养7 d，Western blotting检测脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）和胶质细胞源性神经营养因子（glial cell line-derived neurotrophic factor，GDNF）的表达。用上述4组冷冻保存4周的坐骨神经和SD大鼠新鲜坐骨神经（Fresh组），移植至Wistar大鼠对应坐骨神经10 mm缺损处对其进行修复（分别为37 ℃-Allo组、42 ℃-Allo组、45 ℃-Allo组、Con-Allo组、Fresh-Allo组），并设置Wistar大鼠坐骨神经同系移植组（Fresh-Iso组），每组大鼠 6只。移植术后20周，电生理检测肌肉复合动作电位（compound muscle action potential， CMAP）和神经传导速度（nerve conduction velocity，NCV），甲苯胺蓝染色观察再生有髓神经纤维数，透射电镜观察再生神经超微结构。结果·42 ℃ 组 HSP70表达水平高于37 ℃组、45 ℃组和Con组（均
P<0.05）。冷冻保存4周后42 ℃组MHC-Ⅰ、MHC-Ⅱ表达水平与37 ℃组、45 ℃组、Con组差异均无统计学意义，但42 ℃组caspase-3、caspase-9表达水平较37 ℃组、45 ℃组、Con组降低，坐骨神经细胞存活率较高（均
P<0.05）。培养7 d后，42 ℃组BDNF、GDNF表达水平较37 ℃组、45 ℃组、Con组升高（均
P=0.000）。移植术后20周，42 ℃-Allo组CMAP、NCV、有髓神经纤维数、髓鞘厚度均优于Con-Allo组、Fresh-Allo组（均
P=0.000）。结论·HSP70高表达能减轻大鼠坐骨神经冷冻保存损伤，提高保存后神经细胞活性，促进异体移植后神经再生。
关键词: 热应激预处理, 热休克蛋白70, 冷冻保存损伤, 异体神经移植, 神经再生 Abstract: Objective·To investigate the effect of heat shock protein 70 (HSP70) on cryopreserved rat sciatic nerve cell activity and nerve regeneration after allotransplantation.
Methods·Bilateral sciatic nerves of SD rats were placed in DMEM medium and pretreated with heat stress in vitro at 37 ℃, 42 ℃ and 45 ℃ for 1 h (37 ℃ group, 42 ℃ group and 45 ℃ group, respectively). A fresh nerve group was set up as the control (Con) group. The number of nerves in each group was 36. The expression of HSP70 in sciatic nerve was detected by Western blotting. The previously described nerves were stored in a cryopreservation solution with liquid nitrogen for 4 weeks. The expressions of major histocompatibility complex class Ⅰ (MHC-Ⅰ), MHC-Ⅱ, caspase-3 and caspase-9 were determined by Western blotting. The survival of sciatic nerve cells was detected by flow cytometry. The cryopreserved sciatic nerves were cultured at 37 ℃ with 5% CO2 for 7 d. The expressions of brain-derived neurotrophic factors (BDNF) and glial cell line-derived neurotrophic factors (GDNF) were detected by Western blotting. The cryopreserved sciatic nerves and the fresh sciatic nerves of SD rats were employed to repair the 10-mm-defect of sciatic nerve in the corresponding Wistar rats in the 37 ℃-Allo group, 42 ℃-Allo group, 45 ℃-Allo group, Con-Allo group and Fresh-Allo group. Meanwhile, an isograft group was also established (Fresh-Iso group). Each group had six Wistar rats. After 20 weeks of transplantation, the compound muscle action potential (CMAP) and nerve conduction velocity (NCV) were detected using electrophysiology. Toluidine blue staining was performed to observe the number of regenerated myelinated nerve fibers. The ultrastructure of the regenerated nerves was visualized through a transmission electron microscope.
Results·HSP70 expression level was higher in the 42 °C group than that in the 37 °C group, 45 °C group and Con group, respectively (all P<0.05). After 4 weeks of cryopreservation, the expressions of MHC-Ⅰ and MHC-Ⅱ in the 42 ℃ group were not statistically different from those in the 37 ℃ group, 45 ℃ group and Con group, respectively. The expression levels of caspase-3 and caspase-9 in the 42 °C group were lower than those in the 37 °C group , 45 °C group and Con group, respectively, and the survival rate of sciatic nerve cells was increased (all P<0.05). After 7 d of culture, the expressions of BDNF and GDNF in the 42 ℃ group were higher than those in the 37 ℃ group, 45 ℃ group and Con group (all P=0.000), respectively. After 20 weeks of the transplantation, CMAP, NCV, the number of myelinated nerve fibers and myelin sheath thickness in the 42 °C-Allo group were superior to those in the Cont-Allo group and Fresh-Allo group (all P=0.000).
Conclusion·Highly expressed HSP70 can minimize cryopreservation injury in sciatic nerves of rats, improve nerve cell viability after preservation, and promote nerve regeneration after allotransplantation.
Key words: heat shock pretreatment, heat shock protein 70 (HSP70), cryolesion, nerve allograft, nerve regeneration PDF全文下载地址:
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