去细胞异种角膜基质与去细胞异种结膜基质的免疫原性研究

目的：应用Gal抗原缺失小鼠从体液免疫、细胞免疫、植入物局部组织病理等多方面评价去细胞异种角膜基质（猪源）与去细胞异种结膜基质（猪源）的免疫原性。方法：将用于每种材料的小鼠分为3个实验组：对照组（Con）、原材料组（T1）和基质组（T2）。将去细胞异种角膜基质与去细胞异种结膜基质及其原材料植入经兔血红细胞2次预免疫的Gal抗原缺失小鼠体内，于植入1个月后取材，分别分析2种基质及其原材料组与对照组在小鼠血清总抗体、抗-Gal抗体、细胞因子，脾脏淋巴细胞不同亚型的细胞表面分子表达水平，以及植入物局部组织病理等方面的变化。结果：2种基质及其原材料组在植入1个月后小鼠血清总抗体含量、细胞因子含量与对照组相比均无显著性差异。少数脾脏淋巴细胞表面分子表达水平与对照组相比有显著性差异（P<0.05），但均在对照组历史数据变异范围内。然而，小鼠血清抗gal抗体检测结果显示：去细胞异种结膜基质植入1个月时基质及其原材料组小鼠血清抗-gal抗体水平比对照组显著升高（P<0.05）；而去细胞异种角膜基质植入1个月时基质及其原材料组小鼠血清抗-gal抗体水平与对照组相比均无显著性差异。植入物局部组织病理显示：去细胞异种结膜基质植入1个月时大部分已降解，组织学显示轻微炎症反应，原材料组已无材料残留，组织学显示无明显炎症反应。去细胞异种角膜基质及原材料组植入1个月时植入物未发生明显降解，基质及原材料组的组织学反应均为重度炎症反应。结论：本研究结果证实了Gal抗原缺失小鼠对材料中残留Gal抗原的敏感性，应用Gal抗原小鼠能够科学地评价动物源性生物材料的异种免疫原性风险。

Objective: To evaluate the immunogenicity of acellular xenogeneic corneal matrix (pig sourced)and acellular xenogeneic conjunctival matrix (pig sourced)based on Gal antigen-deficient mice through humoral immunity, cellular immunity and local histopathology. Methods: Mice for each material were divided into three groups:control group (Con), raw material group (T1)and matrix group (T2). Acellular xenogeneic corneal matrix, acellular xenogeneic conjunctival matrix, and their raw materials were implanted into Gal antigen-deficient mice, which were twice pre-immunized with rabbit red blood cells (RRBC). The tissues were collected after 1 month, and the serum total antibody content, serum anti-Gal antibody level, serum cytokines content, levels of spleen lymphocytes cell expressing different surface antigens, and the histopathology of local tissues implanted with materials were analyzed respectively. Results: There was no significant difference in serum total antibody content and cytokine content between both matrix and their raw material groups compared with the control group post 1 month implantation. The expression levels of a few spleen lymphocytes surface antigens were significantly different from those of the control group (P<0.05), but all of them were within the range of historical data variation in the control group without biological significance. For acellular xenogeneic conjunctival matrix, the results of serum anti-gal antibody in the raw material and matrix group were significantly higher than that in the control group post-1 month implantation (P<0.05). However, for acellular xenogeneic corneal matrix, there was no significant difference in serum anti-gal antibody levels in the raw material group or the matrix group compared with the control group post-1 month implantation. The histopathology data showed that most of the materials were degraded in the acellular xenogeneic conjunctival matrix group post-1 month implantation, and accompanied by a slight inflammation, and in the raw material group, materials were completely degraded and there was no obvious inflammation. For acellular xenogeneic corneal matrix, the implants had no obvious degradation, and accompanied by severe inflammation in both matrix and raw material groups post-1 month implantation. Conclusion: The results of this study confirmed the sensitivity of Gal antigen deficient mice to residual Gal antigen in materials, and the xenogeneic immunogenicity risk of animal-derived biomaterials can be scientifically evaluated by using Gal antigen deficient mice.

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