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

doi:10.3969/j.issn.1006-9771.2020.04.007

《中国康复理论与实践》 ›› 2020, Vol. 26 ›› Issue (4): 407-422.doi: 10.3969/j.issn.1006-9771.2020.04.007

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

张松^1,²,黄英如^1,²(),石一峰^1,²,刘云霄^1,²,冼华^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:2019-12-17 修回日期:2020-01-08 出版日期:2020-04-25 发布日期:2020-04-27
通讯作者: 黄英如 E-mail:hyr12678@126.com
作者简介:张松(1987-),男,汉族,重庆市人,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(81373668);国家自然科学基金面上项目(81674002)

Effects of Endogenous Neurotrophic Factors on Nerve Regeneration after Cryopreserved Sciatic Nerve Allograft in Rats

ZHANG Song^1,²,HUANG Ying-ru^1,²(),SHI Yi-feng^1,²,LIU Yun-xiao^1,²,XIAN Hua^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:2019-12-17 Revised:2020-01-08 Published:2020-04-25 Online:2020-04-27
Contact: HUANG Ying-ru E-mail:hyr12678@126.com
Supported by:
National Natural Science Foundation of China (General)(81373668);National Natural Science Foundation of China (General)(81674002)

摘要/Abstract

摘要：

目的探讨内源性神经营养因子(ENTFs)对冷冻保存大鼠坐骨神经同种异体移植后神经再生的影响。方法 15 mm雌性Sprague-Dawley大鼠坐骨神经置于DMEM溶液中,37 ℃、5% CO₂分别体外预处理1 d、3 d、7 d、14 d和21 d (A组、B组、C组、D组和E组),设置新鲜神经对照组(F组)。Western blotting检测神经的胶质细胞源性神经营养因子(GDNF)、神经生长因子(NGF)、Bcl-2、Bax、Caspase-3、主要组织相容性复合体(MHC)-Ⅰ、MHC-Ⅱ蛋白表达。将上述6组神经置于冷冻保存液中液氮保存4周,Calcein-AM/Propidium Iodide染色、激光共聚焦显微镜观察保存后神经活细胞和死细胞情况。用上述冷冻保存4周的坐骨神经和新鲜坐骨神经(G组),同种异体移植修复雌性Wistar大鼠坐骨神经10 mm缺损(A′组、B′组、C′组、D′组、E′组、F′组和G′组),设置同系移植组(H′组)。移植术后1周,免疫荧光染色观察CD₈⁺T细胞、巨噬细胞入侵移植物情况,ELISA法检测受者血清白细胞介素(IL)-2、干扰素(IFN)-γ、肿瘤坏死因子(TNF)-α水平;移植术后20周,电生理检测肌肉复合动作电位(CMAP)和神经传导速度(NCV),称重计算腓肠肌湿重比,神经丝(NF)200免疫荧光染色、甲苯胺蓝染色和透射电镜观察再生神经组织学。结果与F组相比,C组、D组和E组GDNF、NGF蛋白表达均增加(P < 0.05);B~E组Bcl-2蛋白表达降低( P < 0.05),Bax和Caspase-3蛋白表达均增加( P < 0.05);A组~E组MHC-Ⅰ、MHC-Ⅱ蛋白表达均降低( P <0.05)。坐骨神经冷冻保存4周后,与F组和G组相比,C组、D组和E组活细胞数量降低。同种异体移植术后1周,与F′组和G′组相比,C′组、D′组和E′组移植物CD₈⁺T细胞、巨噬细胞减少,受者血清IL-2、TNF-α水平降低(P < 0.05)。移植术后20周,C′组、D′组和E′组CMAP、NCV、腓肠肌湿重比、再生有髓神经纤维数及髓鞘厚度均显著优于F′组和G′组( P <0.05),C′组、D′组和E′组移植神经NF200表达高于F′组和G′组。结论体外预处理大鼠坐骨神经能诱导ENTFs表达,高表达ENTFs的坐骨神经冷冻保存后异体移植能促进受者神经再生和功能恢复。

关键词: 周围神经损伤, 同种异体移植, 周围神经冷冻保存, 内源性神经营养因子, 神经再生, 大鼠

Abstract:

Objective To investigate the effect of endogenous neurotrophic factor (ENTFs) on nerve regeneration after cryopreserved sciatic nerve allograft in rats.Methods The 15-mm sciatic nerves from female Sprague-Dawley rats were placed in DMEM solution and pretreated in vitro for 1 day, 3 days, 7 days, 14 days, and 21 days at 37 ℃ with 5% CO ₂ (groups A, B, C, D and E) respectively. Fresh nerve group (group F) was set up. The protein expression of glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), Bcl-2, Bax, Caspase-3, major histocompatibility complex (MHC)-I and MHC-II was detected by Western blotting. The above six groups were cryopreserved in liquid nitrogen for four weeks. The living cells and dead cells of the nerves after cryopreservation were observed by Calcein-AM/propidium iodide staining. In addition, the above six cryopreserved groups and another fresh nerve group (group G) were transplanted to the Wistar rats by allografting (groups A', B', C', D', E', F' and G'). Isograft group (group H') was set up. One week after transplantation, the expression of CD ₈⁺ T cells and macrophages of the graft were observed by immunofluorescence staining, and the plasma levels of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were detected by ELISA. Twenty weeks after transplantation, the compound muscle action potential (CMAP) and nerve conduction velocity (NCV) were examined by electrophysiology. The wet weight ratio of gastrocnemius muscle was calculated by the operational side compared with the contralateral side. The expression of neurofilament protein (NF) 200 of the transplanted nerves was observed by immunofluorescence staining. The number of myelinated nerve fibers was analyzed by toluidine blue staining. The thickness of myelinated was analyzed by electron microscopy.Results Compared with group F, the protein expression of GDNF and NGF increased in groups C, D and E (P< 0.05); the protein expression of Bcl-2 reduced and the protein expression of Bax and Caspase-3 increased in groups B, C, D, and E (P< 0.05); the protein expression of MHC-I and MHC-II decreased in all the pretreated groups (P< 0.05). Four weeks after cryopreservation, compared with groups F and G, the number of living cells decreased in groups C, D and E. One week after transplantation, compared with groups F' and G', the expression of CD₈⁺ T cells and macrophages decreased, and the plasma concentration of IL-2 and TNF-α decreased in groups C', D' and E' (P<0.05). Twenty weeks after transplantation, CMAP, NCV, the wet weight ratio of gastrocnemius muscle, the number of axon and thickness of myelin sheath were better in groups C', D' and E' than in groups F' and G' (P<0.05), as well as the expression of NF200.Conclusion ENTFs can be induced by pretreating the sciatic nerve in vitro. Cryopreserved sciatic nerve with high expression of ENTFs could promote nerve regeneration and functional recovery after allograft.

Key words: peripheral nerve injury, allograft, peripheral nerve cryopreservation, endogenous neurotrophic factors, nerve regeneration, rats

中图分类号:

R745.4

张松,黄英如,石一峰,刘云霄,冼华. 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.

ZHANG Song,HUANG Ying-ru,SHI Yi-feng,LIU Yun-xiao,XIAN Hua. Effects of Endogenous Neurotrophic Factors on Nerve Regeneration after Cryopreserved Sciatic Nerve Allograft in Rats[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2020, 26(4): 407-422.

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组别	n	GDNF/β-actin	NGF/β-actin
A组	6	0.186±0.026	0.092±0.023
B组	6	0.454±0.032^a	0.251±0.029^a
C组	6	0.937±0.030^a,b	0.864±0.063^a,b
D组	6	1.242±0.041^a,b,c	1.212±0.040^a,b,c,
E组	6	0.982±0.058^a,b,d	0.458±0.042^a,b,c,d
F组	6	0.542±0.025^a,c,d,e	0.265±0.023^a,c,d,e
F值		339.661	358.905
P值		< 0.001	< 0.001

组别	n	Bax/β-actin	Bcl-2/β-actin	Bax/Bcl-2	Caspase-3/β-actin
A组	6	0.203±0.031	1.128±0.066	0.180±0.030	0.255±0.019
B组	6	0.446±0.026^a	1.061±0.038	0.422±0.035	0.528±0.031^a
C组	6	0.754±0.037^a,b	0.894±0.038^a,b	0.843±0.020^a,b,	0.613±0.028^a
D组	6	0.858±0.047^a,b	0.691±0.027^a,b,c	1.244±0.090^a,b,c	0.741±0.035^a,b,c
E组	6	1.001±0.064^a,b,c,d	0.629±0.034^a,b,c	1.597±0.177^a,b,c,d	0.750±0.046^a,b,c
F组	6	0.189±0.043^b,c,d,e	1.267±0.094^b,c,d,e	0.149±0.028^b,c,d,e	0.167±0.030^b,c,d,e
F值		192.279	63.485	148.898	171.621
P值		< 0.001	< 0.001	< 0.001	< 0.001

组别	n	MHC-Ⅰ/GAPDH	MHC-Ⅱ/GAPDH
A组	6	0.311±0.021	0.639±0.033
B组	6	0.154±0.014^a	0.576±0.022
C组	6	0.128±0.010^a	0.467±0.024^a,b
D组	6	0.070±0.006^a,b,c	0.311±0.019^a,b,c
E组	6	0.045±0.004^a,b,c	0.139±0.015^a,b,c,d
F组	6	0.422±0.021^a,b,c,d,e	0.816±0.036^a,b,c,d,e
F值		358.905	349.990
P值		< 0.001	< 0.001

组别	n	IL-2	IFN-γ	TNF-α
A′组	6	44.373±2.598	238.883±14.146	182.373±12.779
B′组	6	43.786±2.604	234.133±19.821	164.290±8.081
C′组	6	39.139±0.780	226.973±12.371	153.900±6.210^a
D′组	6	36.864±2.130^a,b	225.423±11.857	136.743±4.721^a,b
E′组	6	35.121±1.122^a,b	213.220±15.041	128.970±6.730^a,b,c
F′组	6	44.848±1.858^c,d,e	257.103±14.482^e	184.997±2.015^c,d,e
G′组	6	48.575±1.221^c,d,e	338.757±9.551^a,b,c,d,e,f	190.680±4.430^b,c,d,e
H′组	6	35.173±1.721^a,b,f,g	203.947±11.377^f,g	123.757±3.462^a,b,c,f,g
F值		22.218	27.632	45.485
P值		< 0.001	< 0.001	< 0.001

组别	n	CMAP(mA)	NCV(m/s)
A′组	6	0.127±0.031	4.983±0.649
B′组	6	0.202±0.036	5.367±0.592
C′组	6	0.235±0.051^a	6.417±0.436^a
D′组	6	0.432±0.050^a,b,c	8.733±0.622^a,b,c
E′组	6	0.273±0.044^a,d	7.083±0.739^a,b,d
F′组	6	0.108±0.041^b,c,d,e	4.217±0.306^b,c,d,e
G′组	6	0.062±0.025^b,c,d,e	4.067±0.592^b,c,d,e
H′组	6	0.475±0.043^a,b,c,e,f,g	9.117±0.542^a,b,c,e,f,g
F值		80.228	68.753
P值		< 0.001	< 0.001

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

Effects of Endogenous Neurotrophic Factors on Nerve Regeneration after Cryopreserved Sciatic Nerve Allograft in Rats

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组别	n	腓肠肌湿重比
A′组	6	0.523±0.020
B′组	6	0.557±0.021
C′组	6	0.615±0.024^a
D′组	6	0.765±0.031^a,b,c
E′组	6	0.642±0.026^a,b,d
F′组	6	0.484±0.010^c,d,e
G′组	6	0.444±0.022^a,b,c,d,e
H′组	6	0.782±0.039^a,b,c,e,f,g
F值		71.790
P值		< 0.001

组别	n	轴突密度(/mm²)	髓鞘厚度(µm)
A′组	6	11776.524±816.723	0.604±0.060
B′组	6	13302.008±1808.003	0.712±0.057
C′组	6	15970.413±427.301^a	0.749±0.074^a
D′组	6	21256.583±1009.335^a,b,c	0.989±0.058^a,b,c
E′组	6	16877.374±1916.679^a,d	0.764±0.047^a,d
F′组	6	11570.388±1277.176^c,d,e	0.564±0.050^b,c,d,e
G′组	6	7471.276±689.650^a,b,c,d,e,f	0.421±0.034^a,b,c,d,e,f
H′组	6	22674.714±1806.836^a,b,c,e,f,g	1.010±0.096^a,b,c,e,f,g
F值		44.513	76.527
P值		< 0.001	< 0.001

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