Gal抗原缺失小鼠的应用示范：动物源性硬脑膜补片的免疫原性反应评价

目的：采用Gal抗原缺失模式小鼠评价动物源性硬脑膜补片残余免疫原风险，考察Gal抗原缺失模式小鼠的应用价值。方法：在经外源性Gal抗原（兔血红细胞）预免的Gal抗原缺失小鼠皮下分别植入硬脑膜补片（产品）与牛心包（原材料）4周，分析小鼠血清总抗体，特异性抗-Gal抗体，与免疫排斥反应及炎症反应相关的细胞因子表达水平，脾脏淋巴细胞亚型及胸腺、局部病理，综合考察各实验组的差异。结果：牛心包植入组与对照组相比，小鼠血清抗-Gal IgG和抗-Gal IgM水平显著升高（分别为约4倍和约1.7倍）；而硬脑膜补片植入组小鼠血清抗-Gal IgG、抗-Gal IgM水平与对照组相比均无明显差异。除硬脑膜补片植入组小鼠血清IL-4含量下降之外，其余细胞因子包括IL-1β、IL-10、IL-2、IL-6、IL-12P70与IFN-γ含量，血清总IgG、IgM水平在各组间均无显著差异。脾脏淋巴细胞亚型分析显示，硬脑膜补片植入组和心包植入组与对照组相比均无显著性变化。胸腺病理未见异常；局部病理显示硬脑膜补片植入组炎症：Ⅰ-Ⅱ级，纤维化：Ⅰ-Ⅱ级，较心包植入组（炎症：Ⅱ-Ⅲ级，纤维化：Ⅱ-Ⅲ级）反应减轻。研究结果表明动物源性硬脑膜补片产品的Gal抗原介导的免疫原性反应与原材料相比显著降低。然而，硬脑膜补片植入组的植入物局部仍存在一定程度的炎症反应和纤维化现象。结论：硬脑膜补片残余免疫原风险与原材料相比显著降低，与对照组无显著性差异；Gal抗原缺失模式小鼠血清抗-Gal IgG、抗-Gal IgM能够科学地评价动物源性生物材料Gal抗原相关的免疫原性，可以作为动物源性生物材料脱抗原处理工艺有效性的特异性指标之一；提示Gal抗原缺失模式小鼠对动源性生物材料的异种免疫原性风险评价具有极高的应用价值。

Objective:To evaluate the residual immunogenic risks of endocranium patch derived from animal tissues,and to explore the applicability of Gal antigen-deficient mice. Methods:Gal antigen-deficient mice (with rabbit red blood cells pre-immunization) were used as the model animals,endocranium patch (medical device product) and bovine pericardium (raw material for endocranium patch production) were subcutaneously implanted in the model animals for 4 weeks. The index including serum total antibody,specific anti-Gal antibody,level of serum cytokines related to immune rejection and inflammation,spleenic lymphocyte subtype and thymus and local pathology were assessed. Results:The level of anti-Gal IgG and anti-Gal IgM in mice serum in the bovine pericardial implanted group was significantly higher than that in control (about 4 times and 1.7 times,respectively). However,there was no significant change in anti-Gal IgG and anti-Gal IgM in mice serum in endocranium patch implanted group compared with that in control. Except for the decrease of IL-4 in endocranium patch implanted group,there were no significant changes in the level of cytokines including IL-1beta,IL-10,IL-2,IL-6,IL-1 2P70 and IFN-γ,total IgG and IgM in mice serum among each group. There were no significant changes in the bovine pericardium and endocranium patch implanted group in spleenic lymphocytes subtype analysis compared with that in control. The pathology of thymus was not abnormal. Local pathology of implant site in endocranium patch implanted group showed classⅠ-Ⅱ inflammation and classⅠ-Ⅱ fibrosis,which were less severe than those in the bovine pericardium group (classⅡ-Ⅲ inflammation and classⅡ-Ⅲ fibrosis). These results demonstrated that Gal antigen-mediated immunogenic response of animal-derived endocranium patch product is significantly lower than that of raw material (bovine pericardium). However,there was still some degree of inflammation and fibrosis in the implant site of the endocranium patch implant group. Conclusion:The residual Gal antigen-mediated immune response of endocranium patch was significantly lower than that of raw material,and there was no significant difference compared with control group. The level of anti-Gal IgG and anti-Gal IgM can be used to evaluate the Gal antigen-mediated immunogenic response with animal-derived biomaterials. This can also be applied as one of the specific index to assess the effectiveness of antigen-removal treatment during animal-derived biomaterials manufacture processes,suggesting that Gal antigen-deficient mice is a useful tool for evaluation of heterogeneous immunogenicity risk of animal-derived biomaterials.