摘要
目的探讨人/猕猴血清IgG、IgM抗体对不同基因修饰猪外周血单核细胞(PBMC)的细胞毒性,初步筛选适合人/猕猴异种肾移植的猪基因型。方法构建Gal抗原敲除猪(GTKO),Gal抗原和Sda抗原同时被敲除猪(GTKO/β4GalNT2KO),Gal抗原和Neu5Gc抗原同时被敲除猪(GTKO/CMAHKO),以及Gal、Sda和Neu5Gc三种异种抗原同时被敲除猪(GTKO/β4GalNT2KO/CMAHKO,TKO),并在上述基因修饰猪的三种中分别转入人保护性蛋白hCD55(GTKO/hCD55、GTKO/β4GalNT2KO/hCD55和TKO/hCD55)。分离每种基因修饰猪的PBMC进行体外培养。抽取20只猕猴血清,流式细胞术检测血清IgG/IgM抗体与野生型猪(WT)和各种基因修饰猪PBMC的结合情况,结果用几何平均荧光强度(Gmean)表示;同时利用补体依赖的细胞毒性试验(CDC)检测IgG/IgM对不同基因修饰猪PBMC的杀伤作用;抽取80位健康人血清,重复进行上述检测。结果(1)猕猴血清IgM与GTKO/β4GalNT2KO/hCD55型猪PBMC的结合(7.70)显著低于其与WT(45.05)、GTKO/hCD55(33.21)、TKO/hCD55(22.52)猪PBMC的抗体结合,CDC结果与之类似(9.83%vs 97.79%,9.83%vs 42.01%,9.83%vs 47.57%)。(2)人血清IgG/IgM与GTKO/CMAHKO和TKO/hCD55猪PBMC的结合较低,而对TKO/hCD55猪PBMC的杀伤最低。结论GTKO/β4GalNT2KO/hCD55猪可能是目前猪-猕猴异种肾移植的最佳供体猪;而对于临床猪-人异种肾移植而言,导入人“保护性”基因hCD55的TKO猪可能是最佳选择。
Objective To explore the cytotoxicity of human/macaque serum IgG and IgM antibodies on peripheral blood mononuclear cells(PBMC)from different gene-modified porcine to preliminarily screen out the most suitable gene-modified pigs for xenogeneic kidney transplantation of humans or macaques.Methods Theα-Gal gene-knockout(GTKO)pigs,α-Gal/β-1,4N-acetylgalactosaminyltransferase gene-knockout(GTKO/β4GalNT2KO)pigs,α-Gal/cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene knockout(GTKO/CMAHKO)pigs and GTKO/β4GalNT2KO/CMAHKO triple gene knockout(TKO)pigs were constructed.The human protective protein hCD55 was transferred into the three kinds of gene modified pigs respectively.The PBMCs were isolated from each kind of gene-modified pigs.The binding of IgG and IgM antibodies to wild type(WT)pig and various gene-modified pig PBMCs was detected by flow cytometry from 20 macaque sera,the results were expressed by geometric mean fluorescence intensity(Gmean);at the same time,complement dependent cytotoxicity test(CDC)was used to detect the killing effect of IgG and IgM on different gene modified porcine PBMCs.The serum was collected from 80 people,and then the above-mentioned experiments were repeated.Results(1)The binding of serum IgM from macaques to PBMC from GTKO/β4GalNT2KO/hCD55 pigs was significantly weaker than than that to WT,GTKO/hCD55 and TKO/hCD55 pig PBMCs(Gmean:7.70 vs 45.05,7.70 vs 33.21,7.70 vs 22.52),which was consistent with the results of CDC assay(Gmean:9.83%vs 97.79%,9.83%vs 42.01%,9.83%vs 47.57%).(2)The bindings of human serum IgG and IgM to GTKO/CMAHKO and TKO/hCD55 pig PBMCs were low,while the cytotoxic effect of TKO/hCD55 pig PBMCs was the lowest.Conclusion GTKO/β4GalNT2KO/hCD55 pig may be the best donor for pig-macaque xenotransplantation,while TKO pig with the human“protective”gene hCD55 may be the best choice for clinical pig-human xenotransplantation.
作者
江建
李涛
王毅
JIANG Jian;LI Tao;WANG Yi(Department of Organ Transplantation,The Second Affiliated Hospital of Hainan Medical University,Haikou,Hainan 570100,China)
出处
《中国临床研究》
CAS
2023年第1期7-11,共5页
Chinese Journal of Clinical Research
基金
海南省重大科技计划项目(ZDKT2019009)
海南省自然科学基金(821QN413)。
关键词
异种移植
基因修饰猪
猕猴血清
人血清
抗体
外周血单核细胞
免疫球蛋白
细胞毒性
Xenotransplantation
Gene-modified pigs
Macaque serum
Human serum
Antibody
Peripheral blood mononuclear cells
Immunoglobulin
Cell toxicity
