实验动物与比较医学2023，Vol.43Issue(6)：585-594,10.DOI:10.12300/j.issn.1674-5817.2023.065
猪血型PCR鉴定方法的建立
Establishment of PCR Identification Method for Pig Blood Type
摘要
Abstract
Objective Xenotransplantation is an effective way to address the shortage of human organ donors,but it faces serious immune rejection reactions,including hyperacute rejection caused by blood type differences.Establishing a stable,convenient,and reliable method for pig blood type identification can quickly screen suitable donor pigs for xenograft research.Methods Banna miniature inbred pigs,Diannan small eared pigs,and Bama Xiang pigs were selected as the research objects.DNA was extracted from the blood,oral buccal mucosa,and fetal fibroblasts of the three strains of pigs using DNA extraction kits.The target fragment of the ABO homologous gene EAA intron 7 in pigs was amplified using PCR method.Blood agglutination reaction was used to detect hemolysis in pig anterior vena cava whole blood after adding anti A and B antibodies.Immunohistochemical method was used to detect the expression level of A antigen in pig heart,liver,spleen,lung,and kidney tissues.Immunofluorescence method was used to detect the expression level of A antigen in pig oral mucosa.By comparing the results of different methods for determining pig blood types,the stability and reliability of the PCR method were verified,and a convenient PCR based pig blood type identification method was established.Results Firstly,the blood PCR results of 69 inbred strains of Banna miniature pigs,7 Diannan small eared pigs,and 34 Bama Xiang pigs showed 20 AO blood types,66 AA blood types,and 24 O blood types.The PCR results of fetal fibroblasts from 47 Diannan small eared pigs showed that all 47 fetuses were O blood type.Among them,the oral mucosal PCR results of 8 gene edited cloned pigs were consistent with those of donor fetal fibroblasts,all of which were O blood type.The oral mucosal PCR results of 8 wild-type pigs(2 inbred lines of Banna miniature pigs,4 Diannan small eared pigs,and 2 Bama Xiang pigs)were consistent with the blood PCR identification results.Then,11 inbred lines of Banna miniature pigs,4 Diannan small eared pigs,and 2 Bama Xiang pigs were randomly selected for blood agglutination reaction validation,and the results were consistent with the PCR identification results of both blood samples and oral mucosa samples.Moreover,immuno-histochemical analysis was performed on the heart,liver,lung,kidney,and spleen tissues of one Banna miniature pig inbred line and two Bama Xiang pigs,and the results were consistent with blood PCR identification and blood agglutination reaction results.Finally,oral mucosal samples were collected from 2 inbred strains of Banna miniature pigs and 1 Bama Xiang pig for immunofluorescence detection,and the results were consistent with the blood PCR identification results.Conclusion By collecting fetal cells and oral mucosal samples from live pigs for PCR detection,the blood type of pigs can be accurately and efficiently identified,providing a convenient method for blood type screening of xenograft donor pigs.关键词
猪/血型/PCR技术/异种器官移植/血液/胎儿成纤维细胞/口腔黏膜Key words
Pigs/Blood type/PCR technology/Xenotransplantation/Blood/Fetal fibroblasts/Oral mucosa分类
医药卫生引用本文复制引用
王娇祥,王艳,胡珂,徐凯祥,魏太云,角德灵,赵恒,赵红业,魏红江..猪血型PCR鉴定方法的建立[J].实验动物与比较医学,2023,43(6):585-594,10.基金项目
国家重点研发计划干细胞及转化研究重点专项"异种移植用人源化基因编辑供体猪的构建及临床前研究"(2019YFA0110700) （2019YFA0110700）
云南省重大科技专项计划"猪-非人灵长类异种器官移植关键技术的创新与应用"(202102AA310047) （202102AA310047）
云南省创新团队"云南农业大学动物基因编辑和体细胞克隆技术省创新团队(带头人:魏红江)"(2017HC017) （带头人:魏红江）
