热休克蛋白70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

doi:10.3969/j.issn.1674-8115.2021.09.009

摘要/Abstract

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% CO₂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

中图分类号:

R745.4

石一峰, 黄英如, 刘云霄, 张松, 冼华. 热休克蛋白70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[J]. 上海交通大学学报(医学版), 2021, 41(9): 1190-1196.

Yi-feng SHI, Ying-ru HUANG, Yun-xiao LIU, Song ZHANG, Hua XIAN. Effects of heat shock protein 70 on cytoactive of sciatic nerve cryopreservation and nerve regeneration after allograft[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(9): 1190-1196.

图/表 7

图1 坐骨神经HSP70表达Note： A. Western blotting analysis of HSP70. B. Quantitative analysis of HSP70 protein expression in each group.①P=0.001, ②P=0.000, compared with the 42 ℃ group; ③P=0.000, ④P=0.002, compared with the Con group.

Fig 1 Expression of HSP70 in sciatic nerve

图2 坐骨神经MHC-Ⅰ和MHC-Ⅱ表达Note： A. Western blotting analysis of MHC-Ⅰ and MHC-Ⅱ. B. Quantitative analysis of MHC-Ⅰ protein expression in each group. C. Quantitative analysis of MHC-Ⅱ protein expression in each group. ①P=0.001, ②P=0.000, compared with the Fresh group.

Fig 2 Expression of MHC-Ⅰ and MHC-Ⅱ in sciatic nerve

图3 坐骨神经caspase-3和caspase-9表达Note： A. Western blotting analysis of caspase-3 and caspase-9. B. Quantitative analysis of caspase-3 protein expression in each group. C. Quantitative analysis of caspase-9 protein expression in each group. ①P=0.001, ②P=0.000,compared with the 42 ℃ group; ③P=0.001, ④P=0.000,compared with the Con group; ⑤P=0.000, ⑥P=0.001, compared with the Fresh group.

Fig 3 Expression of caspase-3 and caspase-9 in sciatic nerve

图4 坐骨神经细胞存活情况Note：A. Assessment of the survival rates of sciatic nerve cells by flow cytometry after cryopreservation for 4 weeks. B. Quantitative analysis of cell viability. ①P=0.000,compared with the 42 ℃ group; ②P=0.000,compared with the Con group; ③P=0.000,compared with the Fresh group.

Fig 4 Survival of sciatic nerve cells

图5 培养7 d后坐骨神经中BDNF和GDNF的表达Note：A. Western blotting analysis of BDNF and GDNF. B. Quantitative analysis of BDNF protein expression in each group. C. Quantitative analysis of GDNF protein expression in each group. ①P=0.000,compared with the 42℃ group;②P=0.000,compared with the Con group; ③P=0.006, ④P=0.001, compared with the Fresh group.

Fig 5 Expression of BDNF and GDNF in sciatic nerve cultured for 7 d

图6 术后20周各组坐骨神经CMAP检测Note：A. Waveform of CMAP detection. B. Quantitative analysis of CMAP. C. Quantitative analysis of NCV. ①P=0.000, compared with the 42 ℃-Allo group; ②P=0.000, compared with the Con-Allo group; ③P=0.000, compared with the Fresh-Allo group; ④P=0.000, ⑤P=0.001,compared with the Fresh-Iso group.

Fig 6 Detection of CMAP of sciatic nerve in each group at the 20th week after surgery

图7 术后20周再生有髓神经纤维数和再生神经超微结构Note：A. Toluidine blue staining (×400, bar=50 μm). B. Number of myelinated nerve fibers. C. Electron microscopy (×6 000, bar=2 μm). D. The thickness of myelin sheath. ①P=0.000, compared with the 42℃-Allo group; ②P=0.000, compared with the Con-Allo group; ③P=0.000,compared with the Fresh-Allo group; ④P=0.000,compared with the Fresh-Iso group.

Fig 7 Number of myelinated nerve fibers and the ultrastructure of regenerated nerve at the 20th week after surgery

参考文献 20

1	Panagopoulos GN, Megaloikonomos PD, Mavrogenis AF. The present and future for peripheral nerve regeneration[J]. Orthopedics, 2017, 40(1): e141-e156.
2	Qing LM, Chen HW, Tang JY, et al. Exosomes and their microRNA cargo: new players in peripheral nerve regeneration[J]. Neurorehabilit Neural Repair, 2018, 32(9): 765-776.
3	Li Z, Zhang S, Li J, et al. Nerve regeneration in rat peripheral nerve allografts: evaluation of cold-inducible RNA-binding protein in nerve storage and regeneration[J]. J Comp Neurol, 2019, 527(17): 2885-2895.
4	梁玮, 王美霞, 刘宝林. 低温保存对生物样本及其生物大分子的影响[J]. 中国生物医学工程学报, 2017, 36(5): 615-621.
5	Shefa U, Jung J. Comparative study of microarray and experimental data on Schwann cells in peripheral nerve degeneration and regeneration: big data analysis[J]. Neural Regen Res, 2019, 14(6): 1099-1104.
6	马永宾, 葛锁华, 周丹, 等. 大鼠骨髓间充质干细胞促进RSC96细胞凋亡并抑制其增殖和迁移[J]. 细胞与分子免疫学杂志, 2017, 33(2): 190-195.
7	Chebotareva N, Bobkova I, Shilov E. Heat shock proteins and kidney disease: perspectives of HSP therapy[J]. Cell Stress Chaperones, 2017, 22(3): 319-343.
8	贺学岗, 张广智, 马占军, 等. 热休克蛋白在脊髓损伤中的研究进展[J]. 生命科学研究, 2020, 24(4): 339-344.
9	樊欣, 彭仁. 热休克蛋白70: 生物学功能与作用机制研究进展[J]. 生命科学, 2019, 31(3): 270-278.
10	Rosenzweig R, Nillegoda NB, Mayer MP, et al. The Hsp70 chaperone network[J]. Nat Rev Mol Cell Biol, 2019, 20(11): 665-680.
11	Jiang B, Xiao W, Shi Y, et al. Heat shock pretreatment inhibited the release of Smac/DIABLO from mitochondria and apoptosis induced by hydrogen peroxide in cardiomyocytes and C2C12 myogenic cells[J]. Cell Stress Chaperones, 2005, 10(3): 252-262.
12	Leung AM, Redlak MJ, Miller TA. Role of heat shock proteins in oxygen radical-induced gastric apoptosis[J]. J Surg Res, 2015, 193(1): 135-144.
13	Healy DA, Daly PJ, Docherty NG, et al. Heat shock-induced protection of renal proximal tubular epithelial cells from cold storage and rewarming injury[J]. J Am Soc Nephrol, 2006, 17(3): 805-812.
14	Nasouti R, Khaksari M, Mirzaee M, et al. Trehalose protects against spinal cord injury through regulating heat shock proteins 27 and 70 and caspase-3 genes expression[J]. J Basic Clin Physiol Pharmacol, 2020, 31(1). DOI:10.1515/jbcpp-2018-0225.
15	Kim JY, Kim JW, Yenari MA. Heat shock protein signaling in brain ischemia and injury[J]. Neurosci Lett, 2020, 715: 134642.
16	Zhou M, Hu M, He SM, et al. Effects of RSC96 schwann cell-derived exosomes on proliferation, senescence, and apoptosis of dorsal root ganglion cells in vitro[J]. Med Sci Monit, 2018, 24: 7841-7849.
17	Zuo W, Xu F, Zhang K, et al. Proliferation-enhancing effects of gastrodin on RSC96 Schwann cells by regulating ERK1/2 and PI3K signaling pathways[J]. Biomed Pharmacother, 2016, 84: 747-753.
18	肖龙, 黄英如, 李沿江, 等. 二甲基亚砜和乙二醇对玻璃化保存大鼠坐骨神经的协同保护作用[J]. 上海交通大学学报(医学版), 2015, 35(7): 940-946.
19	张松, 黄英如, 石一峰, 等. 内源性神经营养因子促进冷冻保存大鼠坐骨神经异体移植后神经再生的作用[J]. 中国康复理论与实践, 2020, 26(4): 407-422.
20	Wang Y, Zhang S, Li ZJ, et al. The effects of triptolide on the cellular activity of cryopreserved rat sciatic nerves and nerve regeneration after allotransplantation[J]. Int J Neurosci, 2020, 130(1): 83-96.