去细胞同种异体神经种植许旺细胞的体外实验：组织工程化人工神经应用的可行性

韩金豹1，高宏阳2，赵宝辉1，韩久卉1，陈居文3

课题背景：本实验最早酝酿于2002年，是“胎兔雪旺细胞桥接体复合体修复周围神经缺损的实验研究”课题的组成部分。在前期的研究中分别探讨了神经生长因子对胎兔许旺细胞培养的影响以及聚氧乙烯辛烷基酚(TritonX-100)制备去细胞的神经移植体。

应用要点：实验将胎兔许旺细胞作为种子细胞，减少了细胞移植后的免疫排斥反应，在细胞培养过程中加入神经生长因子，获得了大量高纯度的许旺细胞，有效解决了种子细胞问题。实验过程中也观察到胎兔许旺细胞在体外的确能在经去细胞处理的同种异体神经段中生长、繁殖，并沿保留完整的基底膜管纵形排列生长，这些都为下一步去细胞同种异体神经桥接复合体的体内移植提供了很好的研究基础。

同行评价：文章体外实验证实，胎兔坐骨神经培养的许旺细胞能用在成年兔坐骨神经制备的去细胞神经中生长良好，为组织工程化人工神经的构建提供了有效的方法，对研究神经缺损的解决方法具有启发作用。不足之处在于未能探讨许旺细胞与移植物的有机结合过程。�

摘要
目的:许旺细胞作为种子细胞在组织工程化人工神经制备中的作用已被学术界所接受。胎兔的神经系统已发育完善，而免疫系统尚未成熟，探讨将胎兔许旺细胞植入去细胞同种异体神经桥接体制备组织工程化人工神经修复周围神经缺损的可行性。
方法：实验于2005-03/2006-03在河北医科大学第三医院完成。①实验材料：SPF级怀孕28 d的新西兰白兔2只、成年新西兰白兔1只。②实验过程：包括胎兔许旺细胞培养、去细胞神经桥接体的制备以及许旺细胞与去细胞神经桥接体的体外种植3个步骤。使用双酶消化法培养许旺细胞并将其种植于经3% TritonX-100作用96 h后的同种异体神经中。③实验评估：分别于培养的1，3，5 d通过石蜡切片苏木精-伊红染色观察许旺细胞在桥接体中的生长情况。
结果：①采用双差速贴壁法去除绝大部分成纤维细胞后在细胞培养的初期使用阿糖胞苷抑制成纤维细胞生长，后期培养液中加入神经生长因子促进许旺细胞生长能获取大量许旺细胞且纯度可达90%以上。②用3%Triton X-100作用96 h可去除周围神经中的细胞和髓鞘而保留完整的神经基底膜管和纤维支架结构，并且对神经基底膜主要成分－层粘连蛋白无明显影响。③采用胎兔坐骨神经培养的许旺细胞能在用成年兔坐骨神经制备的去细胞桥神经接体中生长良好，并且有迁移成行的特性。
结论：种植许旺细胞重新细胞化的去细胞同种异体神经将可能成为一种理想的组织工程化人工神经。
关键词：周围神经；组织工程；化学萃取；许旺细胞

韩金豹，高宏阳，赵宝辉，韩久卉，陈居文.去细胞同种异体神经种植许旺细胞的体外实验：组织工程化人工神经应用的可行性[J].中国组织工程研究与临床康复，2008，12(11):2006-2010
[www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2006(ps).pdf]

李涛，臧洪敏，唐天驷.1，25-(OH)2VitD3促进组织工程骨的血管化和骨化[J].中国组织工程研究与临床康复，2008，12(11):2001-2005 [www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2001(ps).pdf]

1河北医科大学第三医院手外科，河北省石家庄市 050051；2河北医科大学第一医院骨科，河北省石家庄市 050031； 3天津市第四中心医院骨二科，天津市 300140

韩金豹，男，1952年生，河北省衡水市人，汉族，1978年河北医科大学毕业，教授，主任医师，硕士生导师，主要从事周围神经损伤的研究。
hanjinbao1952 @yahoo.com.cn

中图分类号:R318
文献标识码:A
文章编号:1673-8225
(2008)11-02006-04

收稿日期：2007-08-18
修回日期：2007-10-07
(07-50-8-4495/W·Y)

In vitro study of populating Schwann cell into acellular nerve allograft: Feasibility of applying tissue engineered artificial nerve

Abstract

AIM：It is universally accepted that Schwann cells are the seed cell for preparing tissue engineered artificial nerve. Fetus rabbit nerve system has been developed completely, and immune system is still immature. This study discussed the feasibility of populating fetus rabbit Schwann cells into acellular nerve bridging allograft and preparing tissue engineered artificial nerve to repair the peripheral nerve defects.
METHODS: The experiment was carried out in the Third Hospital of Hebei Medical University between March 2005 and March 2006.①Two New Zealand rabbits of SPF grade, 28-day pregnancy, and one adult New Zealand rabbit.②Experimental managements included three steps: culture of the fetus rabbit Schwann cells, preparation of acellular nerve bridging and in vitro graft of Schwann cells and acellular nerve bridging. Enzyme digestion (trypsin/collagenase) was applied to culture Schwann cells, which were then micro-injected into nerves that had been treated with 3% TritonX-100 for 96 hours.③The paraffin section was sampled at 1, 3, 5 days of culture and the growth of Schwann cells in bridging graft was observed by hematoxylin-eosin staining.
RESULTS: ①We achieved a large amount of high purity (above 90%) Schwann cells by the nerve growth factor, which promoted Schwann cells, added in the culture medium at the latter stage, and double 30-min differential adhesion culture and Arab-c prohibition methods at the early stage of culture were applied to remove fibroblasts.②Schwann cells, axons and myelin sheaths could be successfully removed after treated by 3% Triton X-100 for 96 hours. The basal lamin tubes and collagen fibers structure remained intact. Moreover, the main component of nerve basement membrane-laminin was not damaged.③Schwann cells from fetus rabbit sciatic nerve grew well in the acellular nerve bridge graft of adult rabbit, and migrated in rows.
CONCLUSION: The acellular nerve allografts populated Schwann cells may be an ideal way to prepare tissue-engineered nerve graft.

Han JB, Gao HY, Zhao BH, Han JH, Chen JW. In vitro study of populating Schwann cell into acellular nerve allograft: Feasibility of applying tissue engineered artificial nerve.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(11):2006-2010(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-11/11k-2006(ps).pdf]

1Department of Hand Surgery, Third Hospital of Hebei Medical University, Shijiazhuang 050051, Hebei Province, China; 2Department of Orthopaedics, First Hospital of Hebei Medical University, Shijiazhuang 050031, Hebei Province, China; 3Second Department of Orthopaedics, Fourth Central Hospital of Tianjin, Tianjin 300140, China

Han Jin-bao, Professor, Chief physician, Tutor of master, Department of Orthopaedics, Fourth Central Hospital of Tianjin, Tianjin 300140, China
hanjinbao1952@yahoo.com.cn

Received: 2007-08-18
Accepted: 2007-10-07