探讨基因编辑猪-恒河猴异种心脏移植前后供体猪心组织S100钙结合蛋白A8/A9(S100A8/A9)表达及其与巨噬细胞炎症反应的关系。

分别选取3头巴马基因编辑猪作为供体，3只成年雄性恒河猴作为受体。手术方式为心脏异位移植。应用蛋白质印迹法(WB)检测S100A8/A9、诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)、TNF-α、IL-18、TGF-β和IL-10表达水平。应用免疫组织化学染色检测CD3、CD19和CD68表达水平。正态分布计量资料组间比较采用成组t检验，非正态分布计量资料组间比较采用Wilcoxon符号秩和检验。P<0.05为差异有统计学意义。

供、受体心脏均成功复搏。WB检测结果示移植前后猪心组织S100A8相对表达量分别为(1.000±0.000)和(3.234±0.790)，S100A9相对表达量分别为(1.000±0.000)和(2.796±0.617)，差异均有统计学意义(t＝－4.893和－5.042，P均<0.05)。HE染色结果示移植后基因编辑猪心肌细胞广泛坏死，并可见出血，伴炎性细胞浸润。免疫组织化学染色结果示移植后基因编辑猪心肌组织CD68表达呈阳性。三例受体猴术后外周血单核细胞百分比未见持续升高趋势。猪心组织移植前后iNOS相对表达量分别为(1.000±0.000)和(13.763±0.229)，Arg-1分别为(1.000±0.000)和(0.143±0.071)，差异均有统计学意义(t＝－96.518和20.930，P均<0.05)。移植前猪心组织TNF-α、IL-18、TGF-β、IL-10相对表达量均为(1.000±0.000)，移植后相对表达量分别为(1.447±0.108)、(3.656±0.596)、(0.741±0.034)和(0.682±0.049)，差异均有统计学意义(t＝－7.126、－7.725、13.164和11.178，P均<0.05)。

异种移植后猪心组织S100A8/A9、iNOS及促炎因子表达水平均显著升高，抗炎标志物水平降低，提示S100A8/A9可能通过驱动巨噬细胞M1极化加重异种心脏移植炎症反应。

To investigate the myocardial expression of S100 calcium-binding proteins A8/A9 (S100A8/A9) in gene-edited pig donors before and after heterotopic cardiac xenotransplantation, and to delineate their relationship with macrophage-mediated inflammatory responses.

Three Bama gene-edited pigs were selected as donors and three adult male rhesus macaques served as recipients; heterotopic cardiac xenotransplantation was performed. Western blotting (WB) was employed to quantify the protein levels of S100A8/A9, inducible nitric-oxide synthase (iNOS), arginase-1 (Arg-1), TNF-α, IL-18, TGF-β, and IL-10. Immunohistochemical staining was performed to evaluate the tissue expression of CD3, CD19, and CD68. Between-group comparisons of normally distributed continuous data were performed using the independent-samples t-test, whereas the Wilcoxon signed-rank test was used for non-normally distributed continuous data. A P-value <0.05 was considered statistically significant.

Both donor and recipient hearts were successfully reperfused. WB analysis revealed that the relative expression levels of S100A8 in porcine cardiac tissue before and after transplantation were (1.000±0.000) and (3.234±0.790), respectively, while those of S100A9 were (1.000±0.000) and (2.796±0.617), respectively; the differences were statistically significant (t=-4.893 and -5.042, all P<0.05). HE staining showed extensive cardiomyocyte necrosis, hemorrhage, and inflammatory-cell infiltration in the transplanted gene-edited pig hearts. Immunohistochemical staining showed positive CD68 expression in the myocardial tissue of the gene-edited pig after transplantation. In all three recipient monkeys, the peripheral blood monocyte ratio did not exhibit a sustained postoperative increase. The relative expression levels of iNOS in porcine cardiac tissue before and after transplantation were (1.000±0.000) and (13.763±0.229), respectively, while those of Arg-1 were (1.000±0.000) and (0.143±0.071), respectively; the differences were statistically significant (t=-96.518 and 20.930, all P<0.05). Before transplantation, the relative expression levels of TNF-α, IL-18, TGF-β and IL-10 in porcine cardiac tissue were all (1.000±0.000); and the relative expression levels of them were (1.447±0.108), (3.656±0.596), (0.741±0.034), and (0.682±0.049) after transplantation, with statistically significant differences (t=-7.126, -7.725, 13.164 and 11.178, all P<0.05).

The expression of S100A8/A9, iNOS, and pro-inflammatory cytokines in gene-edited pig donor hearts was markedly up-regulated, whereas anti-inflammatory markers were down-regulated, indicating that S100A8/A9 may exacerbate the inflammatory response in cardiac xenografts by promoting macrophage M1 polarization.