中国组织工程研究

中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (2): 281-285.doi: 10.3969/j.issn.2095-4344.2958

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

α-半乳糖基抗原缺失模型兔的研制与评价

穆钰峰1，2，魏利娜1，吴  勇1，邵安良1，陈  亮1，屈树新2，徐丽明1，2   

  1. 1中国食品药品检定研究院，北京市   102629；2西南交通大学材料科学与工程学院，四川省成都市   611756
  • 收稿日期:2020-01-14 修回日期:2020-01-19 接受日期:2020-03-09 出版日期:2021-01-18 发布日期:2020-11-21
  • 通讯作者: 徐丽明，博士，研究员，中国食品药品检定研究院，北京市 102629；西南交通大学材料科学与工程学院，四川省成都市 611756 屈树新，博士，教授，西南交通大学材料科学与工程学院，四川省成都市 611756
  • 作者简介:穆钰峰，男，1995年生，江苏省连云港市人，汉族，西南交通大学、中国食品药品检定研究院联合培养在读硕士，主要从事生物医学工程、医疗器械生物学评价研究。 魏利娜，1987年生，河南省内黄县人，汉族，2014年北京大学毕业，博士，助理研究员，主要从事医疗器械生物学评价研究。
  • 基金资助:
    国家重点研发计划(2016YFC1103203)

Development and evaluation of alpha-galactosyl antigen-deficient rabbit model

Mu Yufeng1, 2, Wei Lina1, Wu Yong1, Shao Anliang1, Chen Liang1, Qu Shuxin2, Xu Liming1, 2     

  1. 1National Institutes for Food and Drug Control, Beijing 102629, China; 2School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 611756, Sichuan Province, China
  • Received:2020-01-14 Revised:2020-01-19 Accepted:2020-03-09 Online:2021-01-18 Published:2020-11-21
  • Contact: u Liming, MD, Researcher, National Institutes for Food and Drug Control, Beijing 102629, China; School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 611756, Sichuan Province, China Qu Shuxin, PhD, Professor, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 611756, Sichuan Province, China
  • About author:Mu Yufeng, Master candidate, National Institutes for Food and Drug Control, Beijing 102629, China; School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 611756, Sichuan Province, China Wei Lina, MD, Assistant research, National Institutes for Food and Drug Control, Beijing 102629, China
  • Supported by:
     the National Key Research and Development Project of China, No. 2016YFC1103203

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摘要：


文题释义：

Gal抗原：中文全称为α-半乳糖基抗原(α1，3galactosyle，Alpha1，3Gal，或 α-Gal)，是半乳糖基与细胞膜上的蛋白或脂结合构成的一组完全性抗原，研究表明Gal抗原是异种移植中引起超急性免疫排斥反应的主要靶抗原。
CRISPR/Cas9基因编辑技术：是指在sgRNA引导下，利用Cas9核酸酶靶向特定DNA序列并产生双链断裂(DSB)，通过同源重组修复(HDR)或非同源末端连接(NHEJ)的方式，从而实现基因敲除、敲入和染色体转位等。目前，CRISPR/Cas9系统以其快速、简便、高效的特点已经广泛应用于生命科学领域。

背景：有研究表明一种称为α-Gal的糖抗原是动物源性生物材料或异种器官移植引起超急性免疫排斥反应的主要靶抗原。
目的：研制Gal抗原缺失兔模型，预期用于评价植入性医疗器械，其局部植入后对宿主的局部免疫反应及非特异性炎症反应风险。
方法：选用SPF级6-8月龄新西兰大白兔作为模式动物蓝本，制备Gal抗原缺失兔模型。采用CRISPR/Cas9介导的基因编辑技术，针对调控兔子Gal抗原表达的GGTA1基因第8外显子设计构建2条相辅的sgRNA。经转录后将GGTA1 sgRNA mRNAs与Cas9 mRNA共显微注射到体外培养的兔子受精卵中，继续短暂体外培养后植入代孕母兔体内，经自然妊娠获得仔兔。基因编辑成功与否通过凝胶电泳和基因测序进行验证。Gal抗原的表达参照行业标准给出的方法(YY/T 1561-2017)进行检测。研究经中国食品药品检定研究院实验动物资源研究所动物伦理委员会批准[批准号：中检动(福)第2017(B)007号]。
结果与结论：①基因编辑后的胚胎被转移至4只代孕兔体内，自然妊娠后获得15只基因编辑仔兔，仔兔的外观发育及进食行为等未见异常；②凝胶电泳及基因测序结果显示15只仔兔中有14只的靶向基因被成功编辑，但编辑的碱基并不相同；随机检测其主要脏器Gal抗原，表达均降低了99.96%以上；③结果表明，Gal抗原缺失兔子有望用于动物源性医疗器械的免疫原性风险评价和异种骨、角膜等原位植入实验，以便能够更客观科学地评价动物源性医疗器械的安全性和有效性。

https//orcid.org/0000-0001-6059-5681(穆钰峰)；0000-0003-2510-3818(魏利娜)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: Gal抗原, GGTA1基因, CRISPR/Cas9, 基因编辑, 免疫原性, 模式动物, 原位植入, Gal抗原缺失兔子

Abstract:

BACKGROUND: α-Galactosyl (α-Gal) is the main target antigen in hyperacute rejection resulting from animal tissues or xenotransplantation. 
OBJECTIVE: To develop a Gal antigen-deficient rabbit model in order to objectively evaluate the immunogenicity risk of animal-derived biomaterials and the local response to implantation in the host. 
METHODS: New Zealand white rabbits, SPF grade, 6-8 months old, were selected as model animals to prepare Gal antigen-deficient rabbits. Using CRISPR/Cas9-mediated gene editing technology, two complementary sgRNAs were designed and constructed for the 8th exon of GGTA1 gene to regulate Gal antigen expression in rabbits. After transcription, GGTA1 sgRNA mRNAs and Cas9 mRNA were co-microinjected into in vitro cultured rabbit fertilized eggs, which were then implanted into surrogate mother rabbits after a brief in vitro culture, and the neonatal rabbits were obtained by natural pregnancy. The success of gene editing was verified by gel electrophoresis and gene sequencing. The expression of Gal antigen was detected with reference to the method given by the industry standard (YY/T 1561-2017). The study protocol was approved by the Animal Ethics Committee of the Laboratory Animal Resources Laboratory of the National Institutes for Food and Drug Control (approval No. 2017(B)007).
RESULTS AND CONCLUSION: The embryos were transferred to four surrogate rabbits after gene editing. FIfteen gene-edited pups were obtained after natural pregnancy. The appearance and feeding behavior of the pups were not abnormal. Gel electrophoresis and gene sequencing results showed that 14 out of 15 rabbits were successfully edited, but the edited bases were not the same. The Gal antigen of the main organs was detected randomly, and its expression was reduced by more than 99.96%. Therefore, Gal-antigen-deficient rabbits are expected to be used for immunogenic risk assessment of animal-derived medical devices and in situ implantation experiments of heterogeneous bone and cornea, in order to be able to more objectively and scientifically evaluate the safety and effectiveness of animal-derived medical devices.

Key words: Gal antigen, GGTA1 gene, CRISPR/Cas9, gene editing, immune rejection, model animals, orthotopic implantation, Gal antigen-deficient rabbit

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