冷诱导RNA结合蛋白促进冷冻保存大鼠坐骨神经异体移植后神经再生的作用

doi:10.3969/j.issn.1006-9771.2018.04.004

《中国康复理论与实践》 ›› 2018, Vol. 24 ›› Issue (4): 391-400.doi: 10.3969/j.issn.1006-9771.2018.04.004

冷诱导RNA结合蛋白促进冷冻保存大鼠坐骨神经异体移植后神经再生的作用

李子健^1,2,黄英如^1,2,曾欢欢^1,2,汪一^1,2,张松^1,2

1.重庆医科大学中医药学院针灸骨伤教研室，重庆市 400016；
2.中医药防治代谢性疾病重庆市重点实验室，重庆市 400016

收稿日期:2018-02-05 修回日期:2018-03-11 出版日期:2018-04-25 发布日期:2018-04-27
通讯作者: 黄英如。E-mail: hyr12678@126.com
作者简介:李子健(1989-),男,汉族,四川阆中市人,硕士研究生,主要研究方向：周围神经损伤修复。
基金资助:
国家自然科学基金面上项目(No. 81373668; No. 81674002)

Effects of Cold-inducible RNA Binding Protein on Cryopreserved Sciatic Nerve and Nerve Regeneration after Allograft

LI Zi-jian^1,2, HUANG Ying-ru^1,2, ZENG Huan-huan^1,2, WANG Yi^1,2, ZHANG Song^1,2

1. Department of Acupuncture and Orthopaedics of Traditional Chinese Medicine College, Chongqing Medical University, Chongqing 400016, China;
2. Chongqing Key Laboratory of Traditional Chinese Medicine for Prevention and Cure of Metabolic Diseases, Chongqing 400016, China

Received:2018-02-05 Revised:2018-03-11 Published:2018-04-25 Online:2018-04-27
Contact: HUANG Ying-ru. E-mail: hyr12678@126.com
Supported by:
Supported by National Natural Science Foundation of China (General) (No. 81373668; No. 81674002)

摘要/Abstract

摘要： 目的　探讨冷诱导RNA结合蛋白(CIRP)对冷冻保存大鼠坐骨神经活性及同种异体移植后神经再生的影响。方法　将15 mm雄性Sprague-Dawley大鼠坐骨神经片段置于DMEM溶液中，分别在4 ℃、15 ℃、32 ℃预处理24 h (A组、B组、C组)，设置新鲜神经组(D组)。RT-PCR和Western blotting检测CIRP mRNA和蛋白表达。将上述4组神经置于冷冻保存液中液氮保存4周，Calcein-AM/PI荧光染色、激光共聚焦显微镜观察保存后神经活细胞、死细胞情况，Western blotting检测Bax、Bcl-2蛋白表达。取冷冻保存4周的坐骨神经，37 ℃、5% CO2培养7 d，Western blotting检测神经生长因子(NGF)、神经胶质细胞源性神经营养因子(GDNF)蛋白表达；用冷冻保存4周或新鲜雄性Sprague-Dawley大鼠坐骨神经，修复对应的雄性Wistar大鼠坐骨神经10 mm缺损(A′组、B′组、C′组、D′组、E′组)，设立同系移植新鲜组(F′组)。术后4周，免疫组化染色观察移植段神经CD4+T淋巴细胞入侵，ELISA法检测血清白细胞介素(IL)-6、干扰素(IFN)-γ水平；术后20周，电生理检测肌肉复合动作电位(CMAP)和运动神经传导速度(MNCV)，甲苯胺蓝染色分析再生有髓神经纤维数目和髓鞘厚度，电镜观察再生神经超微结构。结果　C组神经CIRP mRNA及蛋白表达均高于A组和B组(P<0.05)。冷冻保存4周后，与A组、B组、D组相比，C组神经活细胞数量较多，Bax表达降低(P<0.05)，Bcl-2表达升高(P<0.05)；C组冷冻保存神经分泌NGF、GDNF表达均高于A组和B组(P<0.05)。同种异体移植术后4周，与E′组相比，C′组CD4+ T淋巴细胞减少，血清IL-6、IFN-γ水平降低(P<0.05)，但C′组与D′组和F′组比较无显著性差异(P>0.05)。术后20周，C′组CMAP、MNCV、再生神经轴突密度及髓鞘厚度均优于A′、B′、D′组和E′组(P<0.05)。再生神经超微结构显示，与A′组、B′组、D′组和E′组相比，C′、F′组有髓神经纤维数量多，粗细均匀，分布广泛，髓鞘厚。结论　32 ℃预处理可促进坐骨神经CIRP高表达，CIRP高表达对冷冻保存大鼠坐骨神经具有保护作用，能维持保存后神经的生物活性，促进同种异体移植后受体神经再生。

关键词: 周围神经损伤, 冷诱导RNA结合蛋白, 冷冻保存, 同种异体移植, 神经再生

Abstract: Objective To investigate the effect of cold-inducible RNA binding protein (CIRP) on the viability of cryopreserved sciatic nerve and nerve regeneration after allograft.Methods Sciatic nerve segments of 15 mm from male Sprague-Dawley rats were placed in DMEM solution and pretreated with 4 ℃, 15 ℃ and 32 ℃ for 24 hours (group A, group B and group C, respectively). Fresh nerve group (group D) without pretreatment was set up. The mRNA and protein level of CIRP was detected by RT-PCR and Western blotting, respectively. The above nerves were cryopreserved in liquid nitrogen for four weeks. The viable cells of the nerve segments after cryopreservation were observed by calcein-AM/propidium iodide staining. The expression of Bax and Bcl-2 was detected by Western blotting. After cryopreservation, the nerve segments were cultured in vitro for one week, the protein level of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) was detected by Western blotting. In addition, the above four cryopreserved groups were transplanted to the Wistar rats by allografting (groups A', B', C' and D'). Fresh nerve allograft group (E') and isograft group (F') were set up. At four-week post operation, the expression of CD4 of the nerve and plasma level of interleukin (IL)-6 and interferon (IFN)-γ were detected by immunohistochemistry and ELISA, respectively. At 20-week postoperation, the muscle compound action potential (CMAP) and motor nerve conduction velocity (MNCV) were examined by electrophysiological examination. The number and the thickness of myelinated nerve fibers were analyzed by toluidine blue staining. The ultrastructure of the sciatic nerve was observed by electron microscopy. Results The mRNA and protein of CIRP were significantly higher in group C than in groups A and B (P<0.05). After 4 weeks of cryopreservation, compared with groups A, B and D, the viable cells increased, the expression of Bax decreased and the expression of Bcl-2 increased in group C (P<0.05). The expression of NGF and GDNF increased in group C than in groups A and B (P<0.05). At four-week postoperation, the expression of CD4 and plasma concentration of IL-6 and IFN-γ significantly decreased in group C' than in group E' (P<0.05), however, no significant difference was found in group C' compared with groups D' and F' (P>0.05). At 20-week postoperation, CMAP, MNCV, the number of axon, and thickness of myelin sheath were significantly better in group C' than in groups A', B', D' and E' (P<0.05). Compared with groups A', B', D' and E', the myelinated nerve fibers were more, the fiber thickness was more uniform, the fiber distribution was wider, and the myelin sheath was thicker in groups C' and F'.Conclusion CIRP was induced at 32 ℃ in the sciatic nerve, which exerted a significant protective effect on the viability of the nerves during cryopreservation, and promoted nerve regeneration and functional recovery after transplantation.

Key words: cold-inducible RNA binding protein, peripheral nerve injury, cryopreservation, allograft, nerve regeneration

中图分类号:

R745

李子健,黄英如,曾欢欢,汪一,张松. 冷诱导RNA结合蛋白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》, 2018, 24(4): 391-400.

LI Zi-jian, HUANG Ying-ru, ZENG Huan-huan, WANG Yi, ZHANG Song. Effects of Cold-inducible RNA Binding Protein on Cryopreserved Sciatic Nerve and Nerve Regeneration after Allograft[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2018, 24(4): 391-400.

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冷诱导RNA结合蛋白促进冷冻保存大鼠坐骨神经异体移植后神经再生的作用

Effects of Cold-inducible RNA Binding Protein on Cryopreserved Sciatic Nerve and Nerve Regeneration after Allograft

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