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摘要:
目的 探讨FAM234A是否为一种新的非Gal异种移植抗原。 方法 用α-1, 3-半乳糖基转移酶基因敲除(GTKO)小型猪的原代猪主动脉内皮细胞(PAEC)免疫食蟹猴。将免疫的猴血清与PAEC孵育, 其中经免疫产生的猴抗猪细胞抗体能够识别并结合PAEC表面的未知抗原。采用流式细胞术测定PAEC表面结合的猴抗猪细胞抗体的平均荧光强度(MFI), 用以表示PAEC表面抗原的含量。利用慢病毒介导的短发夹核糖核酸(shRNA)敲减PAEC中的FAM234A基因, 检测该细胞结合的猴抗猪细胞抗体含量是否减少, 以确定该基因是否为潜在的移植抗原。 结果 PAEC中的非Gal抗原能够在猴体内引发大量的猴抗猪细胞抗体。在GTKO小型猪的PAEC中用shRNA敲减FAM234A基因后, PAEC结合的猴IgG抗体减少。 结论 FAM234A是一种新的非Gal异种移植抗原。 -
关键词:
- 异种移植 /
- 非Gal抗原 /
- 主动脉内皮细胞(PAEC) /
- FAM234A /
- 食蟹猴 /
- α-1, 3-半乳糖基转移酶基因敲除(GTKO) /
- 流式细胞术 /
- 短发夹核糖核酸(shRNA) /
- 基因敲减 /
- 巴马小型猪 /
- 五指山小型猪 /
- 慢病毒
Abstract:Objective To investigate whether FAM234A is a novel non-Gal antigen in xenotransplantation. Methods Cynomolgus monkeys were immunized with primary porcine aorta endothelial cells (PAEC) from α-1, 3-galactosyltransferase gene-knockout (GTKO) minipigs.The serum samples of immunized monkeys were incubated with PAEC.After immunization, the monkey anti-pig cell antibodies were able to recognize and bind with the unknown antigens on the PAEC surface.Flow cytometry was adopted to determine the mean fluorescence intensity (MFI) of monkey anti-pig cell antibodies binding with the PAEC surface to characterize the content of PAEC surface antigen.Lentivirus-mediated short hairpin ribonucleic acid (shRNA) was utilized to knock down the FAM234A gene in PAEC to detect whether the content of the cell-bound monkey anti-pig cell antibody was reduced and determine if this gene was a potential transplant antigen. Results The non-Gal antigen in PAEC could trigger a large quantity of monkey anti-pig cell antibodies in monkeys.After knock down of the FAM234A gene with shRNA in the PAEC of the GTKO miniature pigs, the monkey IgG antibody binding with PAEC was declined. Conclusions FAM234A is a novel non-Gal antigen in xenotransplantation. -
图 1 不同品系GTKO小型猪的PAEC与经免疫的猴血清孵育后结合的猴IgG的含量分析
A图为GTKO五指山小型猪; B图为GTKO巴马小型猪; 红、蓝、黄分别为未孵育血清的PAEC样本、孵育未免疫猴血清的PAEC样本、孵育经PAEC免疫猴血清的PAEC样本
Figure 1. IgG binding of PAECs from different GTKO minipig strains incubated withPAEC-immunized monkey serum
图 2 不同物种FAM234A蛋白的一级序列比对
A图为猪与人序列比对结果; B图为食蟹猴与人序列比对结果; 蓝色为一致序列, 红色为不一致序列
Figure 2. Primary sequence alignment of FAM234A protein in different species
图 3 敲减FAM234A使PAEC与免疫猴血清中的IgG结合减少
A图为五指山小型猪; B图为巴马小型猪; 左侧为抗猪细胞抗体的流式细胞图, 中间为流式细胞参数图, 右侧为不同样本结合抗体的MFI及FAM234A mRNA水平的比较。红色为未孵育血清的PAEC样本, 蓝色为孵育经PAEC免疫猴血清的对照PAEC样本(plko), 黄色为孵育经PAEC免疫猴血清的敲减FAM234A的PAEC样本(plko-FAM234A)
Figure 3. Knock down of FAM234A in PAECs results in a reduction in binding of serum IgG fromimmunized monkey
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