化学去细胞法对粗大神经质量评价方法及影响因素的探讨

目的 通过对粗大神经去细胞方法的研究，探讨对去细胞神经评价和制备的适宜方法。方法 取杂种犬3只，切取5段直径6mm的新鲜坐骨神经，每段长50mm，在3％ Triton-100和7％脱氧胆酸钠溶液中去细胞处理。实验分为5组：Ⅰ组，去细胞2遍；Ⅱ组，去细胞3遍；Ⅲ组，去细胞4遍；Ⅳ组，神经段两端用4号缝合线结扎后去细胞2遍；Ⅴ组，神经段两端用4号缝合线结扎后去细胞3遍。另设对照为未经处理的新鲜犬坐骨神经。从去细胞程度、基底膜板层素（Laminin）活性、脱髓鞘程度及神经纤维管道完整性4个方面进行评价。结果 所有实验组神经段去细胞完全，Laminin活性保存完好。髓鞘染色评分以Ⅳ组和Ⅴ组最低，纤维管道完整性评分以Ⅰ组和Ⅳ组最佳。综合评价对于粗大神经的去细胞处理方法以Ⅳ组处理方法最佳。结论 化学去细胞的神经脱髓鞘程度与去细胞次数不平行。在去细胞神经的质量控制中，应考虑基底膜Laminin的活性、去细胞、脱髓鞘及结构完整性的程度。对于粗大神经的处理，结扎神经段两端后再行去细胞处理可能是一种较好的方法。

QUALITY ESTIMATION AND INFLUENCE FACTORS OF THE LARGER CHEMICALLY ACELLULAR NERVE ALLOGRAFTS IN VITRO

OBJECTIVE: To investigate the preparing procedures for the larger chemically acellular nerve allografts (CANA) and to establish an evaluation criteria and the preparation method. METHODS: The sciatic nerves of the dogs were exposed by a muscle-splitting incision and were isolated free of the underlying fascia. The 50-mm-long segments of the nerve were made. The proximal and distal ends of the nerve segments were labelled and stabilized by pinning the ends to a thin plastic support, and then they were treated according to the following decellularization processes: The nerve segments were rinsed with the distilled water for 9 h, transferred in a 3% Triton-100 solution for 12 h, soaked in 7% sodium deoxycholate for 12 h, and washed in the distilled water for 6 h. All the decellularization steps were performed at room temperature. The nerve segments were divided into 5 subgroups. In Group I, Group II and Group III, the nerve segments were decellularized for 2, 3 and 4 times, respectively. In Group IV and Group V, the two ends of the nerve segments were ligated with a silk line and were decellularized for 2 and 3 times, respectively. Each nerve segment was subdivided into 5 portions from the proximal end to the distal end. The degrees of decellularization, activity of Laminin, degrees of demyelination, and integrity of the nerve fiber tube were observed under microscope and were assessed by a scoring system. RESULTS: In all the groups the activity of Laminin was present and the degrees of decellularization were complete. As for the demyelination of the nerve segments, the myelin sheath in Groups I, II and III was partially preserved, but it completely disappeared in Groups IV and V. The structure of the nerve fiber tube in Groups I and IV was almost normal. The total score for the degrees of decellularization, demyelination, and structural integrity was the lowest in Group IV but the quality was the best. CONCLUSION: The degrees of demyelination are not parallel to the times of decellularization processes. In the quality control of CANA, we should consider the following 4 factors: activity of Laminin, degrees of decellularization, demyelination, and structural integrity. For the larger CANA, ligation of the two ends of the nerve segments during the decellularization procedure may be a better method of ensuring the quality of the decellularization.