人源性同种异体去细胞外周神经材料标准制备方法的研究

Analytical study of human allogenic acellular peripheral nerve in standard preparation methods

目的对目前文献报道使用的5种不同方法制备的人源性同种异体去细胞外周神经材料进行综合分析比较其优劣，以确定可供临床使用的标准的制备流程。方法将人源性神经按5种不同方法进行化学萃取，制备的外周神经支架材料分别行苏木精-伊红（HE）染色、免疫组化（S-100、ColⅠ）和透射电镜、氮含量测定等检测，观察5种方法去除许旺细胞、髓鞘和轴突等抗原成分以及基底膜保存完好的情况。结果分别萃取2次持续24h组神经的HE染色显示去除细胞和轴突彻底，纵切片上未见任何细胞，红染的神经内膜呈波浪状纵形排列，轴突、髓鞘结构消失而形成管柱状空隙；S-100染色呈阴性；ColⅠ染色结果可以看出其结构呈松散不规则的棕黄色结构，而其它处理组则结构相对整齐的纵向带状结构。透射电镜显示该组与各处理之间髓鞘去除差异无统计学意义。氮含量测定显示该组蛋白含量比值最低。结论使用TritonX-100处理24h再分别脱氧胆酸钠24h持续萃取2次，可作为供临床使用的制备人源性去细胞同种异体神经支架材料的标准流程。

Objective To analyze five kinds of allogenic acellular peripheral nerve by different methods and try to establish a standard method for preparing nerve materials. Methods Five kinds of nerve material prepared by different chemical extractions according to nowaday articles were examined by HE staining. Immunohistochemistry and protein ratio was studied by allogenic nerves by virtue of Kjeldahl method in order to know the efficiency of these methods in removal of SCs axons and integrality of the basilar membrane. Results Myelin sheath and cytoblast in group 2 that nerves were extracted by Triton X-100 and Sodium deoxycholate consecutively twice were completely removed, which was well demonstrated in HE staining. Perineurium in red staining were arranged wave-like longitudinally, axons and myelin sheath were replaced by column-like spacing. Col Ⅰ staining were positive in all groups, structure of basilar membrane became loose slightly in the first and second group, and the structure of the other groups were relatively regular. Group 1 and 2 were negative in S-100 staining. There was no difference between group 2 and group 1,3,4 and 5 in sheath removal demonstrated by TEM. Protein ratio in group 2 was the lowest in the measurement with Kjeldahl method. Conclusion The method used in group 2 that nerves were extracted by Triton X-100 and Sodium deoxycholate consecutively twice was the best in allogenic acellular peripheral nerve preparations.