脱细胞脊髓天然支架的制备及形态学观察

Preparation of acellular scaffold of natural spinal cord and observation of morphology

[目的]采用化学脱细胞方法去除细胞和髓鞘成分制作细胞外基质支架,为桥接脊髓损伤缺损提供理想的天然神经支架。[方法]取SD大鼠胸段脊髓约2cm,运用冻融+化学萃取(3%脱氧胆酸钠和1KU/mlDNaseI、RNaseA)的组织工程学方法处理大鼠脊髓组织,并对处理后的脊髓支架分别进行组织学检查,了解脱细胞情况及细胞外基质支架形态。[结果]经过脱细胞处理后,光镜下HE染色脊髓横断面呈网状结构,未见细胞成分存留,纵切面上呈互相交错的管状通路;未见轴突、髓鞘和细胞核。髓鞘染色可见髓鞘脱除彻底,未见髓鞘成分。[结论]本实验采用冻融+化学萃取的组织工程学方法可制备出理想的天然脊髓支架,该支架与脊髓三维组织结构具有高度相似性,有望作为脊髓损伤后桥接物和神经组织工程种子细胞的支架,本实验结果显示3%脱氧胆酸钠和1KU/mlDNa-seI、RNaseA进行脱细胞处理两次是较为合适的,能较为彻底去除细胞及髓鞘成分并较为完整地保留细胞外基质的三维结构。

[Objective] Using a procedure of chemical agent to remove the cells and myethin in spinal cord of rat and to prepare the scaffold of extracellular matrix, so as to obtain an ideal natural spinal cord scaffold to bridge the nerve gap. [Method] Rat spinal cord was cut and treated using the method of freeze thawing and chemical extraction （3% sodiumdeoxycholate and 1KU/ml DNaseI, RNaseA）. Histology was exploited to evaluate the degree of acellular and the structure of the spinal cord scaffold. [Result] In cross section, network of the extracellular matrix was presented in the scaffold. The cells, myethin and axons disappeared after the spinal cord was treated with sodium deoxycholate and DNaseI, RNaseA. Typical network of empty tubes were viewed in longitudinal sections. [Conclusion] An ideal spinal cord scaffold can be produced with the method designed in author＇s experiment. This scaffold has similar three dimensional structure with normal spinal cord, which can be used as a graft to bridge the nerve gap directly or as a scaffold to implant the seeding cells in spinal cord tissue engineering. The experiment indicates that cells and myethin can be removed and the three dimensional structure be reserved by chemical extraction with 3% sodium deoxycholate and 1KU/ml DNaseI, RNaseA. Chemical extraction is an ideal method to prepare tissue engineer scaffold of spinal cord.