中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2018, Vol. 13 ›› Issue (9): 1643-1649.doi: 10.4103/1673-5374.237138

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

-80 ℃冻存同种异体脱细胞神经移植物的多种变化

  

  • 收稿日期:2018-06-15 出版日期:2018-09-15 发布日期:2018-09-15
  • 基金资助:

    中国国家自然科学基金项目（81201546），广东省自然科学基金博士启动项目（2017A030310302），广东省医科科研基金项目（A2016018），广东省科技项目（2016A010103012, 2013B010404019）

Various changes in cryopreserved acellular nerve allografts at −80°C

Yan-Yan Huang1, Xiao-Lu Xu2, Xi-Jun Huang3, Jiang-Hui Liu3, 4, Jian Qi3, Shuang Zhu5, Zhao-Wei Zhu1, Bo He3, Qing-Tang Zhu3, Yang-Bin Xu1, Li-Qiang Gu3, Xiao-Lin Liu3   

  1. 1 Department of Plastic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China
    2 Yale School of Medicine, New Haven, CT, USA
    3 Department of Orthopedic and Microsurgery, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China
    4 Department of Emergency, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China
    5 Department of Joint and Orthopedic, Orthopedic Center, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong Province, China
  • Received:2018-06-15 Online:2018-09-15 Published:2018-09-15
  • Contact: Zhao-Wei Zhu, M.D., Ph.D. or Bo He, M.D., Ph.D.,nmtmemoir@aliyun.com or hebodoc@aliyun.com
  • Supported by:

    This study was supported by the National Natural Science Foundation of China, No. 81201546; the Doctoral Start-up Program of Natural Science Foundation of Guangdong Province of China, No. 2017A030310302; the Medical Scientific Research Foundation of Guangdong Province of China, No. A2016018; grants from the Science and Technology Project of Guangdong Province of China, No. 2016A010103012, 2013B010404019.

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摘要：

实验设计多角度、多方位评价同种异体脱细胞神经在冻存后的组织学、机械性能和生物特性，为长期保存提供依据。将取自人和大鼠的脱细胞同种异体神经于-80℃含10％胎牛血清、10％DMSO和5％蔗糖的RPMI1640培养基中保存1年，然后在40或37℃解冻8min。对人脱细胞同种异体神经通过拉伸测试测量断裂力，以其培养液培养L-929测试细胞存活率。将大鼠脱细胞同种异体神经移植于左侧坐骨神经缺失约15mm的大鼠模型中，以免疫组化染色检测移植物中神经丝200的免疫阳性反应，以HE染色检测腓肠肌中相对肌肉面积，以电镜观察移植物的超微结构变化。-80℃冷冻保存的人脱细胞同种异体神经形态、力学性能及细胞毒性无明显变化；-80℃冻存脱细胞同种异体神经移植的坐骨神经损伤模型大鼠的移植物中神经丝200免疫阳性反应和髓鞘厚度以及腓肠肌萎缩程度与非冻存的同种异体神经移植修复的大鼠接近。表明冻存不会改变脱细胞同种异体神经移植物的生物学相关特性。且可以极大地延长其存储时间。

orcid:0000-0002-9690-4527(Bo He)

 

关键词: 脱细胞同种异体神经移植物, 冻存, 存储, 移植周围, 神经, 神经再生

Abstract:

The experimental design evaluated histological, mechanical, and biological properties of allogeneic decellularized nerves after cryopreservation in a multi-angle, multi-directional manner to provide evidence for long-term preservation. Acellular nerve allografts from human and rats were cryopreserved in a cryoprotectant (10% fetal bovine serum, 10% dimethyl sulfoxide, and 5% sucrose in RPMI1640 medium) at −80°C for 1 year, followed by thawing at 40°C or 37°C for 8 minutes. The breaking force of acellular nerve allografts was measured using a tensile test. Cell survival was determined using L-929 cell suspensions. Acellular nerve allografts were transplanted into a rat model with loss of a 15-mm segment of the left sciatic nerve. Immunohistochemistry staining was used to measure neurofilament 200 expression. Hematoxylin-eosin staining was utilized to detect relative muscle area in gastrocnemius muscle. Electron microscopy was applied to observe changes in allograft ultrastructure. There was no obvious change in morphological appearance or ultrastructure, breaking force, or cytotoxicity of human acellular nerve allografts after cryopreservation at −80°C. Moreover, there was no remarkable change in neurofilament 200 expression, myelin sheath thickness, or muscle atrophy when fresh or cryopreserved rat acellular nerve allografts were applied to repair nerve injury in rats. These results suggest that cryopreservation can greatly extend the storage duration of acellular nerve tissue allografts without concomitant alteration of the physiochemical and biological properties of the engineered tissue to be used for transplantation.

Key words: nerve regeneration, acellular nerve allografts, cryopreservation, storage, transplantation, nerve, neural regeneration