The xenogeneic decellularized corneal matrix (DCM) was expected to be used in lamellar keratoplasty in clinic as the substitute of allogeneic cornea. After decellularization treatment, the remaining risk of xenograft rejection needed to be assessed. The galactose-α1,3-galactose, as the most abundant and closely rejection-related xenogeneic antigen, should be one of the important factors concerned in immunological evaluation. In this study, residual αGal in the DCM was first determined by an enzyme-linked immunosorbent assay method with qualified accuracy and specificity. Then the DCM was implanted subcutaneously into the α1,3-galactosyltransferase gene-knockout (GTKO) mice, accompanied by the implantation in the wild-type C57BL/6 mice as a comparison. The total serum antibody levels, anti-Gal antibody levels, inflammatory cytokines and ratios of splenic lymphocyte subtypes were detected and the histopathological analysis of implants were performed to systematically evaluate the immune responses. The experimental result showed the fresh porcine corneal matrix samples had (9.90 ± 1.54) × 10¹² αGal epitope per mg while the content of residual αGal in the DCM was (7.90 ± 2.00) × 10¹² epitope per mg. The GTKO mice had similar potential of reaction to immune stimulation to that of wild-type C57BL/6 mice. At 4 weeks after implantation of DCM, in WT mice and GTKO mice there were both innate immunity response to the DCM characterized by macrophage infiltration. But the elevations of anti-Gal IgG level and the percentage of splenic natural killer cells were only detected in GTKO mice. These changes were thought to be pertinent to the residual αGal antigen, which could not be detected in WT mice. No further αGal antibody-mediated cellular immunity and significant changes of serum cytokine contents were found in GTKO mice, which perhaps suggested that the immune reactions to the DCM after 4 weeks of implantation were moderate and had minor effect on the survival of the corneal graft.

中文翻译：

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使用GTKO小鼠在异种脱细胞角膜中残留αGal的免疫风险评估。

异种脱细胞角膜基质（DCM）有望在临床中用于层状角膜移植，以替代异体角膜。脱细胞处理后，需要评估异种移植排斥的剩余风险。半乳糖-α1,3-半乳糖是最丰富，与排斥反应最密切相关的异种抗原，应成为免疫学评估的重要因素之一。在这项研究中，DCM中的残留αGal首先通过酶联免疫吸附测定法进行了测定，其准确性和特异性均得到了验证。然后将DCM皮下植入α1,3-半乳糖基转移酶基因敲除（GTKO）小鼠中，同时将其植入野生型C57BL / 6小鼠中进行比较。总血清抗体水平，抗Gal抗体水平，检测炎症细胞因子和脾淋巴细胞亚型的比率，并进行植入物的组织病理学分析以系统评估免疫反应。实验结果表明，新鲜猪角膜基质样品的（9.90±1.54）×10¹² αGal表位每毫克而残留αGal在DCM中的含量为（7.90±2.00）×10 ¹²每毫克表位。GTKO小鼠对免疫刺激的反应潜力与野生型C57BL / 6小鼠相似。DCM植入后第4周，WT小鼠和GTKO小鼠均出现了以巨噬细胞浸润为特征的对DCM的先天免疫应答。但是仅在GTKO小鼠中检测到抗Gal IgG水平的升高和脾自然杀伤细胞的百分比。这些变化被认为与残留的αGal抗原有关，在WT小鼠中无法检测到。在GTKO小鼠中未发现进一步的αGal抗体介导的细胞免疫和血清细胞因子含量的显着变化，这可能表明植入4周后对DCM的免疫反应是中等的，对角膜移植物的存活影响较小。