器官移植2025，Vol.16Issue(4)：526-537,12.DOI:10.12464/j.issn.1674-7445.2025091
GTKO猪的生产及猪-恒河猴肾脏异种移植
Production of GTKO pigs and kidney xenotransplantation from pigs to rhesus macaques
摘要
Abstract
Objective To explore the construction of α-1,3-galactosyltransferase(GGTA1)gene-knockout(GTKO)Diannan miniature pigs and the kidney xenotransplantation from pigs to rhesus macaques,and to assess the effectiveness of GTKO pigs.Methods The GTKO Diannan miniature pigs were constructed using the CRISPR/Cas9 gene-editing system and somatic cell cloning technology.The phenotype of GTKO pigs was verified through polymerase chain reaction,Sanger sequencing and immunofluorescence staining.Flow cytometry was used to detect antigen-antibody(IgM)binding and complement-dependent cytotoxicity.Kidney xenotransplantation was performed from GTKO pigs to rhesus macaques.The humoral immunity,cellular immunity,coagulation and physiological indicators of the recipient monkeys were monitored.The function and pathological changes of the transplanted kidneys were analyzed using ultrasonography,hematoxylin-eosin staining,immunohistochemical staining and immunofluorescence staining.Results Single-guide RNA(sgRNA)targeting exon 4 of the GGTA1 gene in Diannan miniature pigs was designed.The pGL3-GGTA1-sgRNA1-GFP vector was transfected into fetal fibroblasts of Diannan miniature pigs.After puromycin selection,two cell clones,C59#and C89#,were identified as GGTA1 gene-knockout clones.These clones were expanded to form cell lines,which were used as donor cells for somatic cell nuclear transfer.The reconstructed embryos were transferred into the oviducts of trihybrid surrogate sows,resulting in 13 fetal pigs.Among them,fetuses F04 and F11 exhibited biallelic mutations in the GGTA1 gene,and F04 had a normal karyotype.Using this GTKO fetal pig for recloning and transferring the reconstructed embryos into the oviducts of trihybrid surrogate sows,seven surviving piglets were obtained,all of which did not express α-Gal epitope.The binding of IgM from the serum of rhesus monkey 20#to GTKO pig PBMC was reduced,and the survival rate of GTKO pig PBMC in the complement-dependent cytotoxicity assay was higher than that of wild-type pig.GTKO pig kidneys were harvested and perfused until completely white.After the left kidney of the recipient monkey was removed,the pig kidney was heterotopically transplanted.Following vascular anastomosis and blood flow restoration,the pig kidney rapidly turned pink without hyperacute rejection(HAR).Urine appeared in the ureter 6 minutes later,indicating successful kidney transplantation.The right kidney of the recipient was then removed.Seven days after transplantation,the transplanted kidney had good blood flow,the recipient monkey's serum creatinine level was stable,and serum potassium and cystatin C levels were effectively controlled,although they increased 10 days after transplantation.Seven days after transplantation,the levels of white blood cells,lymphocytes,monocytes and eosinophils in the recipient monkey increased,while platelet count and fibrinogen levels decreased.The activated partial thromboplastin time,thrombin time and prothrombin time remained relatively stable but later showed an upward trend.The recipient monkey survived for 10 days.At autopsy,the transplanted kidney was found to be congested,swollen and necrotic,with a small amount of IgG deposition in the renal tissue,and a large amount of IgM,complement C3c and C4d deposition,as well as CD68+macrophage infiltration.Conclusions The kidneys of GTKO Diannan miniature pigs may maintain normal renal function for a certain period in rhesus macaques and effectively overcome HAR,confirming the effectiveness of GTKO pigs for xenotransplantation.关键词
GGTA1基因敲除猪/恒河猴/肾脏异种移植/基因修饰/超急性排斥反应/免疫抑制/外周血单个核细胞/体细胞核移植Key words
GGTA1 gene-knockout pig/Rhesus macaque/Kidney xenotransplantation/Genetic modification/Hyperacute rejection/Immunosuppression/Peripheral blood mononuclear cell/Somatic cell nuclear transfer分类
医药卫生引用本文复制引用
王艳,赵红芳,张雄,朱飞艳,成文敏,赵红业,徐凯祥,Muhammad Ameen Jamal,王振迪,魏红江,常悦,杨畅,魏太云,霍晓颖,陈博威,王娇祥,赵恒,郭建雄..GTKO猪的生产及猪-恒河猴肾脏异种移植[J].器官移植,2025,16(4):526-537,12.基金项目
国家重点研发计划(2019YFA0110700) （2019YFA0110700）
云南省重大科技专项(202102AA310047) （202102AA310047）
云南省人才联合培养计划-青年科学家项目(202303AM140030) （202303AM140030）
