低免疫原性脱细胞神经基质的制备及其理化性能

doi:10.3969/j.issn.2095-9400.2020.06.001

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摘要目的：制备低免疫原性异种脱细胞神经基质并评估其理化性能。方法：以α-1，3-半乳糖苷转移酶基因敲除（GTKO）猪坐骨神经为实验原料对基因敲除猪坐骨神经进行脱细胞处理制备低免疫原性异种脱细胞神经基质，通过HE、DAPI染色及DNA残留量检测评价神经组织的脱细胞效果；I型胶原、层黏连蛋白、纤连蛋白免疫组织化学及天狼猩红染色评价脱细胞神经细胞外基质组分保留情况；α-1，3-半乳糖基（a-gal）免疫荧光检测异种抗原a-gal的残留；扫描电镜（SEM）分析脱细胞神经三维组织结构及孔隙率。结果：组织染色显示脱细胞神经基质内膜、外膜、束膜中的细胞均去除干净，DNA定量结果显示DNA含量下降了95%。天狼猩红染色及免疫组织化学结果显示脱细胞神经组织较好地保留了胶原纤维与细胞外基质主要蛋白。SEM结果显示脱细胞神经较好地保留了其三维组织结构，细胞毒性检测表明脱细胞神经基质无细胞毒性。结论：本研究自主设计的四联脱细胞方案能彻底去除神经组织中的细胞成分与异种抗原，并且较完整地保留了神经组织结构，与天然神经具有高度相似性。

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	赵子建

关键词 ：脱细胞, 低免疫原性, 细胞外基质, 神经移植物, 基因敲除

Abstract：Objective: To prepare low immunogenic xenogeneic decellularized nerve matrix and evaluate its biological properties. Methods: To prepare low immunogenic xenogeneic decellularized nerve matrix by decellularizing the α-1, 3-galactoside transferase gene-knockout (GTKO) pig sciatic nerve with a self-designed combination protocol of hypotonic solution, trypsin, detergent, and nuclease. The decellularization was evaluated by HE, DAPI staining, and the amount of residual DNA. Immunohistochemical staining of type-I collagen, laminin, fibronectin, and Sirius scar staining were used to assess the retention of extracellular matrix components of acellular nerve. A-gal immunofluorescence was used to detect the residual of the heterologous antigen, a-gal. Finally, the three-dimensional (3D) structure and porosity of acellular nerves were analyzed by scanning electron microscopy (SEM). Results: DNA content in the decellularized nerve tissue was 95% lower than that in the natural nerve tissue, and there were few cellular components in the endoneurium, perineurium, and epineurium of acellular nerve. Sirius scar staining and immunohistochemistry showed that the decellularized nerve tissue retained collagen fibers and major extracellular matrix proteins. SEM results showed that the acellular nerve retained its three-dimensional (3D) structure. Cytotoxicity tests showed that the acellular nerve matrix was not cytotoxic. Conclusion: The self-designed decellularization protocol of this study can completely remove the cellular components and heterologous antigens in the nervous tissue, and retain the neural tissue structure more completely. With high similarity to the natural nerves, it has great potential for application as nerve grafts in the future.

Key words： decellularization low immunogenicity extracellular matrix nerve grafts gene knockout

收稿日期: 2019-09-20

基金资助:国家自然科学基金资助项目（81630021）；广东省创新研究团队计划项目（2016ZT06Y432）；温州市领军型创新团队项目（TY2016002）。

通讯作者: 赵子建，教授，博士生导师，Email：azzhao@gdut.edu.cn。

作者简介: 王建英（1994-），女，山西朔州人，硕士生。

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https://xb.wmu.edu.cn/CN/10.3969/j.issn.2095-9400.2020.06.001 或 https://xb.wmu.edu.cn/CN/Y2020/V50/I6/431

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