人类去细胞同种异体神经移植物化学萃取方法的研究

Acellular nerve allograft by chemical extraction in humans

目的 新鲜同种异体神经免疫排斥反应的标靶是细胞、髓鞘。发展新的化学处理方法 ,清除人类周围神经中的细胞和髓鞘 ,萃取粗大和长段的去细胞神经移植物。 方法 切取年轻男性遗体捐献者的长段尺神经 ,以tritonX 10 0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、髓鞘染色及纤维素染色 ,以观察细胞、髓鞘及神经基底膜 ;免疫组织化学染色以显示许旺细胞基底板层 ;行半薄切片及超薄切片透射电镜检查 ,观察超微结构。 结果 去细胞神经的延展性及神经外膜的韧弹性良好。细胞和髓鞘被彻底清除 ,神经基底膜被保留 ;许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。 结论 TritonX 10 0及脱氧胆酸钠化学处理方法 ,可有效清除人类周围神经中的细胞及髓鞘 ,萃取粗大和长段的去细胞神经 ;该神经移植物保持了网管柱状组织结构 ,保留了许旺细胞基底板层及板层素。

Objective To develop a procedure by which Schwann cells and myelin in the peripheral nerve could be removed while the basal lamina tubes remained intact, and to obtain a thick and long acellular nerve allograft in humans. Methods Four ulnar nerves 10.0 cm long and 4.0-5.0 mm in diameter were excised from a donated male body and cleaned from external debris. The nerves were treated with a solution of Triton X-100 and a solution of sodium deoxycholate at room temperature. After a final wash in water, the nerves were stored in phosphate-buffered saline (PBS, pH 7.2) at 4℃. HE, luxol fast blue and fibrin staining were performed to visualize cells, myelin and basal membranes respectively and immunohistochemical staining was performed to visualize the presence of laminin, a Schwann cell lamina component, both in fresh and acellular nerve segments. To reveal overall structure better, methylene blue-fuchsin staining was performed in semithin section. The ultrastructure of acellular and fresh nerves were observed and photographed in a transmission electron microscope.Results The acellular human ulnar nerve was white long cylinder with well elasticity and ductility. HE, myelin and fibrin staining revealed that cells, axons and myelin sheath were removed and basal membrane was preserved after extraction procedure. Staining for the presence of laminin showed that the Schwann cell basal lamina component were present in the nerves after chemical treatment. Methylene blue-fuchsin staining and transmission electron microscopy showed that the myelin sheaths were absent in the extracted nerve segments and empty basal lamina tubes remained in the endoneurium. Conclusions We developed an extracted procedure with the detergents of Triton X-100 and deoxycholate, by which cells, axons and myelin sheaths could be removed from a human ulnar nerve while the basal lamina tubes remain intact and a thick long acellular nerve allograft is obtained. The laminin, a Schwann cell basal lamina component, can be preserved in the acellular nerve.