去细胞同种异体神经支架种植许旺细胞的体外培养

摘要背景：前期的研究中分别探讨了使用复合酶消化法及差速贴壁法对许旺细胞培养的影响以及聚氧乙烯辛烷基酚(Triton X-100)制备同种异体神经支架。目的：通过化学萃取法制备同种异体神经支架，并将培养的许旺细胞与支架进行体外结合培养。方法：取Wistar大鼠双侧坐骨神经，运用30 g/L三硝基甲苯和40 g/L脱氧胆酸钠分别萃取1，2和3次，在萃取神经和未萃取神经的中段取材，行苏木精-伊红染色，S-100及Laminin免疫组织化学染色及透射电镜检测。取SD胎鼠坐骨神经及臂从神经，使用复合酶消化，差速贴壁及Arab-c抑制成纤维细胞生长的培养方法获得大量高纯度的许旺细胞。再把许旺细胞注入同种异体神经支架内，并行透射电镜及扫描电镜观察。 结果与结论：用三硝基甲苯和脱氧胆酸钠萃取后，坐骨神经内细胞和髓鞘被清除，神经基底膜被保留。电镜下可见萃取后的神经由空的神经基底膜管和管之间的胶原纤维构成。萃取次数增加后，神经内残留的S-100蛋白显著减少，但反复萃取后支架结构受破坏。去细胞同种异体神经支架适合许旺细胞体外生长，并有迁移排成行的特性。结果提示，用三硝基甲苯和脱氧胆酸钠萃取2次可去除细胞而保留神经基底膜管，是制备具有仿生结构神经支架的理想方法。神经支架种植许旺细胞后可能成为一种理想的神经缺损修复材料。

In vitro culture of Schwann cells on acellular allogenic nerve scaffold

AbstractBACKGROUND: Previous study has investigate the influence of compound enzyme digestion and differential adherence method on culture of Schwann cells, and alkylphenol polyoxyethylene (Triton X-100) preparation for allogenic nerve scaffold.OBJECTIVE: To prepare an allogenic nerve scaffold using chemical extraction method and culture Schwann cells with the scaffold in vitro. METHODS: The bilateral sciatic nerves of Wistar rats were treated with 30 g/L trinitrotoluene and 40 g/L sodium deoxycholate for extractions. The middle piece of the extracted samples and the un-extracted samples was harvested for hematoxylin-eosin staining, S-100 and laminin immunohistochemical staining, as well as transmission electron microscope observation. Trypsin and collagenase were used to separate Schwann cells from the double sciatic nerves and brachial plexus of SD fetal rats. Highly purified Schwann cells were achieved with differential adherence and Arab-c to eliminate fibroblast. Finally the Schwann cells were injected into the acellular nerve scaffold and the consequence studies were performed by transmission electron microscope and scanning electron microscope. RESULTS AND CONCLUSION: The cells and myelin sheath were removed and basal membrane component was preserved in the sciatic nerve after extraction procedure of trinitrotoluene and sodium deoxycholate. The electron microscopy showed that the extracted nerves are composed of empty basal lamina tubes and collagen fibers between tubes. The residual S-100 protein in nerves was significantly reduced along with the increasing times of extraction, scaffold structure was destroyed after repeated extractions. Acellular allogenic nerve scaffold fits for the growth of Schwann cells, and can transform to aline in vitro. Chemical extraction that uses the detergents of Triton X-100 and deoxycholate is an ideal method to prepare nerve scaffold with biomimetic structure, by the method cells can be removed from the sciatic nerve of Wistar rats while the basal lamina component preserved in the acellular nerve. Allogenous nerve scaffold populated Schwann cells may be an ideal substance to repair the nerve defect after injuries.