GGTA1基因敲除猪去细胞神经移植物制备及其异种移植免疫排斥反应初步研究  

Preliminary study on preparation of decellularized nerve grafts from GGTA1 geneedited pigs and their immune rejection in xenotransplantation

作　　者：刘宇立 赵金娟 宋翔宇 贾志博 李超超 张铁元 李香玲 闫石 何瑞超 彭江[2] LIU Yuli;ZHAO Jinjuan;SONG Xiangyu;JIA Zhibo;LI Chaochao;ZHANG Tieyuan;LI Xiangling;YAN Shi;HE Ruichao;PENG Jiang(Graduate School,Guizhou Medical University,Guiyang Guizhou,550005,P.R.China;Institute of Orthopedics,General Hospital of Chinese PLA,Beijing,100853,P.R.China)

机构地区：[1]贵州医科大学研究生院,贵阳550005 [2]中国人民解放军总医院骨科研究所,北京100853

出　　处：《中国修复重建外科杂志》2025年第2期224-229,共6页Chinese Journal of Reparative and Reconstructive Surgery

摘　　要：目的制备α-1,3-半乳糖转移酶(alpha-1,3-galactosyltransferase,GGTA1)基因敲除猪去细胞神经移植物,初步探讨其用于异种移植的生物相容性。方法无菌条件下获取野生猪、GGTA1基因敲除猪坐骨神经行去细胞处理。采用IB4荧光染色以及ELISA法检测α-半乳糖抗原(alpha-galactosidase,α-gal)含量,以鉴定基因敲除猪坐骨神经GGTA1基因敲除情况,以人坐骨神经作对照;HE染色及扫描电镜观察神经结构;免疫荧光染色和DNA含量测定评价神经去细胞程度。于14只裸鼠脊柱两侧制备皮下囊,各取7只分别植入野生猪、基因敲除猪去细胞神经,1、3、5、7 d后抽血行中性粒细胞计数。结果基因敲除猪神经IB4荧光染色及ELISA检测示GGTA1基因已基本成功敲除。基因敲除猪去细胞神经HE染色示神经结构保存良好,扫描电镜下可见神经内膜管状结构,免疫荧光染色示未见细胞核且保留了天然神经细胞外基质成分,DNA含量较正常神经显著降低(P<0.05)。体内实验示术后1、3、7 d两组中性粒细胞数相似,差异无统计学意义(P>0.05);术后5 d时,基因敲除猪去细胞神经中性粒细胞数低于野生猪,差异有统计学意义(P<0.05)。结论研究制备的GGTA1基因敲除猪去细胞神经移植物,其神经结构保存良好、去细胞完全、保留了天然神经内膜管状结构和成分,体外实验初步验证异种移植后免疫排斥反应低。Objective To prepare decellularized nerve grafts from alpha-1,3-galactosyltransferase(GGTA1)gene-edited pigs and explore their biocompatibility for xenotransplantation.Methods The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization.The alpha-galactosidase(α-gal)content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene,and using human sciatic nerve as a control.HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples.Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples.Fourteen nude mice were taken,and subcutaneous capsules were prepared on both sides of the spine.Decellularized nerve samples of wild-type pigs(n=7)and GGTA1 gene-edited pigs(n=7)were randomly implanted in the subcutaneous capsules.Blood was drawn at 1,3,5,and 7 days after implantation to detect neutrophil counting.Results IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs.HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved;the nerve basement membrane tube structure was visible under scanning electron microscopy;no cell nuclei was observed,and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining;and the DNA content was significantly reduced when compared with the normal nerves(P<0.05).In vivo experiments showed that the number of neutrophils in the two groups were similar at 1,3,and 7 days after implantation,with no significant difference(P>0.05);only at 5 days,the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs(P<0.05).Conclusion The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preser

关 键 词：GGTA1基因敲除猪 周围神经 修复 去细胞基质 异种移植 

分 类 号：R745[医药卫生—神经病学与精神病学]