异种去细胞组织工程化神经支架的制备与形态学研究

The manufacture of accellular tissue engineered nerve xenograft and morphological study

目的:研究新的化学萃取方法,清除成年兔坐骨神经中的细胞和髓鞘,建立成年兔坐骨神经的化学萃取方法,获得去细胞异种神经体移植物。方法:切取健康成年兔双侧坐骨神经,以Triton X-100和脱氧胆酸钠溶液按一定浓度和程序进行化学处理。萃取神经及新鲜神经行HE染色及电镜观察细胞、髓鞘及神经基底膜;免疫组织化学染色显示雪旺细胞基底板层,并进行组织相容性试验。结果:去细胞神经的延展性及神经外膜的韧性和弹性良好,细胞和髓鞘被彻底清除,神经基底膜被保留,雪旺细胞基底板层保留完好,去细胞神经为一种没有细胞髓鞘及其碎片的空的神经基质管,植入的临近部位无急性炎症反应,有较好的生物相容性,无脓肿形成及组织坏死发生。结论:Triton X-100及脱氧胆酸钠化学处理方法,可有效清除周围神经中的细胞及髓鞘,萃取长段的去细胞神经,神经移植物保持了网管柱状组织结构,保留了雪旺细胞板层素,该方法可能是制备具有仿生结构的组织工程化神经支架较为理想的方法。

Objective：To develop a procedure by which cells and myelin in nerve could be removed while the basal lamina tubes remained intact , and to obtain a thick and long acellular nerve xenograft in rabbit. To look for an ideal substance to repair large gap of nerve defect after injuries by culture . Method： Four peripheral nerves were excised from rabbit. The nerves were treated with a solution of Triton X-100 and a solution of sodium deoxyeholate at 4℃ temperature. After a final wash in water, the nerves were stored in phosphate-buffered saline （ PBS , pH 7.2） at 4℃. HE , fibrin staining were performed to visualize cells. Result： Cells, axons and myelin sheath were removed and Schwarm cell basal lamina waspreserved after extraction procedure. Transmission microscopy showed that the myelin sheaths were removed and empty basal lamina tubes remained in the endoneurium. Conclusion：The study developed an extracted procedure, by which cells, axons and myelin sheaths can be removed from a rabbit sciatic nerve while the basal lamina tubes remained intact and a acellular nerve heterogeneity is obtained.