摘要
目的异种器官移植是解决人类器官供体短缺的有效途径,但其面临严重的免疫排斥反应,包括血型差异导致的超急性排斥反应。建立稳定、便捷、可靠的猪血型鉴定方法,可快速筛选合适血型的供体猪用于异种器官移植研究。方法选择版纳微型猪近交系、滇南小耳猪、巴马香猪为研究对象,用DNA提取试剂盒提取3种品系猪的血液、口腔颊黏膜和胎儿成纤维细胞中DNA,通过PCR法对猪ABO血型同源基因EAA第7号内含子附近的目标片段进行扩增。采用血液凝集反应检测猪前腔静脉全血加入抗A、B抗体后的溶血现象,采用免疫组织化学法检测猪心、肝、脾、肺、肾组织中A抗原表达水平,采用免疫荧光法检测猪口腔黏膜中A抗原表达水平。通过对比不同方法测定猪血型的结果,验证PCR方法的稳定性和可靠性,以此建立一种基于PCR的便捷式猪血型鉴定方法。结果首先,69头版纳微型猪近交系、7头滇南小耳猪和34头巴马香猪的血液PCR结果显示AO血型20头,AA血型66头,OO血型24头。47头滇南小耳猪的胎儿成纤维细胞PCR结果显示47个胎儿均为OO血型,其中8头基因编辑克隆猪的口腔黏膜PCR结果与供体胎儿成纤维细胞的PCR结果一致,均为OO血型;8头野生型猪(2头版纳微型猪近交系、4头滇南小耳猪和2头巴马香猪)的口腔黏膜PCR结果均与血液PCR鉴定结果一致。然后,从中随机挑选11头版纳微型猪近交系、4头滇南小耳猪和2头巴马香猪进行血液凝集反应验证,结果均与血液及口腔黏膜PCR鉴定结果一致。而且,对1头版纳微型猪近交系和2头巴马香猪的心、肝、肺、肾、脾组织进行免疫组织化学分析,结果与血液PCR鉴定和血液凝集反应结果相一致。最后,采集其中2头版纳微型猪近交系和1头巴马香猪的口腔黏膜样本进行免疫荧光检测,结果与血液PCR鉴定结果一致。结论通过采集胎儿细胞和活体猪的口腔黏�
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 ran
作者
王娇祥
王艳
胡珂
徐凯祥
魏太云
角德灵
赵恒
赵红业
魏红江
WANG Jiaoxiang;WANG Yan;HU Ke;XU Kaixiang;WEI Taiyun;JIAO Deling;ZHAO Heng;ZHAO Hongye;WEI HongJiang(Key Laboratory of Animal Gene Editing and Animal Cloning in Yunnan Province,Kunming 650201,China;Xenotransplantation Engineering Research Center in Yunnan Province,Kunming 650201,China;College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China;College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China)
出处
《实验动物与比较医学》
CAS
2023年第6期585-594,共10页
Laboratory Animal and Comparative Medicine
基金
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)
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)
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)。
