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  

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

作　　者：韩金豹[1] 高宏阳[2] 赵宝辉[1] 韩久卉[1] 陈居文[3] 

机构地区：[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

出　　处：《中国组织工程研究与临床康复》2008年第11期2006-2010,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

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28d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96h后的同种异体神经中。③实验评估:分别于培养的1,3,5d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%TritonX-100作用96h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。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 carded 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 gr

关 键 词：周围神经 组织工程 化学萃取 许旺细胞 

分 类 号：R318[医药卫生—生物医学工程]