周围神经组织工程载体去细胞神经基膜管的免疫原性研究

Immunogenicity of acellular nerve basal membrane conduit as a vector in the peripheral nerve tissue engineering

目的:为周围神经组织工程中载体的去细胞神经基膜管进行免疫学方面的半定量研究及其进一步的应用提供理论基础。方法:实验于2001-06/2003-03在蚌埠医学院附属医院中心实验室与蚌埠医学院病理学教研室进行并顺利完成。①将18只成年SD大鼠的左侧坐骨神经于坐骨大切迹下稍下方锐性切断其胫神经束,近端结扎,远端任其自由变性。②术后2周,再次手术,切取大鼠左侧远段预变性胫神经和右侧相同部位正常新鲜神经组织段,分别长约30mm。③将预变性和新鲜的神经组织段分别放入相同浓度(5g/L)的十二烷基磺酸钠溶液中进行化学萃取处理,分别得到一种没有细胞及细胞碎片的、空的神经基膜管,即预变性的去细胞神经基膜管和未预变性的去细胞神经基膜管。④分别就预变性神经组、新鲜神经组、预变性去细胞神经基膜管组和未预变性去细胞神经基膜管组进行一般观察、HE染色、锇酸染色、经层粘连蛋白和主要组织相容性复合体抗原II标记的免疫组织化学观察、计算机图像和透射电镜等方法分析。结果:①经过十二烷基磺酸钠萃取的预变性和未预变性神经组织,均基本能够彻底地去除细胞、髓鞘、轴突及其碎片,成为中空的、基底膜结构较完整去细胞基膜管。②经过十二烷基磺酸钠萃取的预变性和未预变性神经组织保留有大量的层粘连蛋白,前者阳性率变化均值为25.8±2.9,后者为13.9±1.8,二者统计学差异具有显著性(P<0.05),其中前者具有优越性。③经过十二烷基磺酸钠萃取的预变性和未预变性神经组织中的主要组织相容性复合体抗原Ⅱ的阳性率极低,同萃取前相比,统计学差异具有显著性(P<0.01)。结论:经过化学萃取后的去细胞未预变性和预变性神经基膜管结构保留均较完整,免疫原性低,可作为组织工程中神经载体,其中预变性组更具有优越性。

AIM： Semi-quantitative study on the immunology of acellular nerve basal membrane conduit of vector in the peripheral nerve tissue engineering is studied, so as to provide theoretical basis for further application. METHODS： This experiment was conducted at the Central Laboratory of the Affiliated Hospital of Bengbu Medical College and the Department of Pathology of Bangbu Medical College between June 2001 and March 2003. ①Tibial nerve tract was sharply cut near from gross cut trace of left sciatic nerve of 18 adult SD rats , ligating proximal part and freeing the distal part. ② 2 weeks after operation, operation was performed again , distal initial denaturative tibial nerve at the left side and normal fresh nerve tissue at the same position on the right side of the rats were cut , with 30 mm in length , respectively. ③ Initial denaturative and fresh nerve tissue segments were put in the sodium dodecylsulphate solution of the same concentration （5 g/L） respectively for chemical extraction management, and initial denaturative blank acellular nerve basal membrane conduit without cells and cell fragments and non-denaturative acellular nerve basal membrane conduit were obtained respectively. ④Some procedures were performed in the initial denaturative nerve group, fresh nerve group, initial denaturative acellular nerve basal membrane conduit group and noninitial denaturative acellular nerve basal membrane conduit group, they were general observation, haematoxylin and eosin staining, osmic acid staining, immunohistechenical staining labeled by laminin and main tissue compatibility complex antigen Ⅱ, computer image analysis and observation under transmission electron miscroscope. RESULTS： ① Initial denaturative tissue and non-initial denaturative nerve tissue extracted by sodium dodecylsulphate both can remove cells, myelin sheath, axon and its fragments and became hollow and acellular nerve basal membrane conduit with complete structure in basal membrane. ②Initial denaturative tissue and non-initial denaturative nerve tissue extracted by sodium dodecylsulphate reserved a lot of laminin, and the positive change rate of the former was 25.8±2.9, and that of the latter was 13.9±1.8,with sigfiificant statistical difference （P 〈 0.05） ③ The positive rate of main tissue compatibility complex antigen Ⅱ in the initial denaturative tissue and non-initial dens iurative nerve tissue extracted by Sodium dodecylsulphate was extremely low as compared with that before extraction, with significant statistical difference （P 〈 0.01 ）. CONCLUSION： The structures of initial denaturative and non-initial denaturative acellular nerve basal membrane conduit are both relatively complete with low immunogenicity after extraction, can be used as nerve vector in the tissue engineering, and initial denaturative group has superiority.