2种Gal抗原缺失小鼠的免疫学特性比较研究

目的：考察2种Gal抗原缺失小鼠对外来抗原刺激的免疫应答特性，分析其用于Gal抗原阳性材料免疫毒性评价的敏感性。方法：采用外来Gal抗原（兔血红细胞，RRBC）免疫刺激2种Gal抗原缺失小鼠（GGTA1单基因敲除，KO与GGTA1/iGb3S双基因敲除，DKO），比较分析经免疫刺激后2种小鼠的免疫应答特性和敏感性，包括：特异性抗-Gal抗体水平，与免疫排斥反应及炎症反应相关的细胞因子表达水平和脾脏淋巴细胞亚型分型比例。结果：GGTA1/iGb3S DKO小鼠经腹腔内RRBC免疫刺激2次后，血清抗-Gal IgG和抗-Gal IgM水平与未接受免疫刺激的小鼠相比，分别升高约41倍和184倍；同时，脾脏T细胞亚型标志物（CD3+CD8+）水平轻度增高。GGTA1 KO小鼠经RRBC免疫刺激2次后，血清抗-Gal IgG和抗-Gal IgM水平与未接受免疫刺激小鼠相比，分别升高约92倍和407倍；同时，脾脏B细胞亚型标志物（CD3-CD19+）水平轻度升高。然而，与GGTA1/iGb3S DKO小鼠相比，可能由于个体间差异较大，除了2次免疫后抗-GalIgG在800倍稀释，抗-Gal IgM在3 200倍稀释时略高之外，其他并不存在显著性差异。2种Gal抗原缺失小鼠在RRBC免疫刺激后的NK细胞亚型标志物水平及细胞因子（IFN-gama，IL-4与IL-12P70）表达水平未见明显变化。结论：2种Gal抗原缺失小鼠均对外来抗原刺激具有较强的敏感性，表现为特异性抗-Gal抗体的显著升高，然而，GGTA1/iGb3S DKO小鼠与GGTA1 KO小鼠相比，未表现出更高的敏感性。因此，作为异种免疫原的免疫学评价模型，GGTA1 KO小鼠和GGTA1/iGb3S DKO小鼠都是敏感的实验动物模型。本研究采用的RRBC预免疫方法既能够提升小鼠的抗-Gal抗体水平，又不引起显著的免疫毒性反应，为后期用于动物源性生物材料的免疫学评价实验动物模型的应用奠定了基础。

Objective:To compare the immune response on foreign antigen stimulation between two types of Gal antigen deficient mice,and analyze their sensitivity for immunotoxicity evaluation of Gal antigen positive materials. Methods:Rabbit red blood cells (RRBC) were used as the foreign Gal antigen to stimulate two types of Gal antigen-deficient mice (GGTA1 gene knockout mice,KO and GGTA1/iGb3S double gene knockout mice,DKO). The characteristics and sensitivity of immune responses between two types of mice were investigated,including the level of specific anti-Gal antibody,the expression level of immune-rejection and inflammatory-response related cytokines and the ratio of splenic lymphocyte subtypes. Results:Compared with the GGTA1/iGb3S DKO mice without RRBC administration,the level of serum anti-Gal IgG and anti-Gal IgM in GGTA1/iGb3S DKO mice with RRBC administration (after two treatments) increased by 41 times and by 184 times,respectively. The level of spleen T cell subtype marker (CD3+CD8+) was slightly increased. Compared with the GGTA1 KO mice without RRBC administration,serum anti-Gal IgG and anti-Gal IgM in GGTA1 KO mice with RRBC administration (after two treatments) increased by 92 times and by 407 times,respectively. The level of spleen B cell subtype marker (CD3-CD19+) was slightly increased. However,compared to GGTA1/iGb3S DKO mice,excluding the fact that anti-Gal IgG (dilution of 800 times) and anti-Gal IgM (dilution of 3 200 times) were slightly higher,there was no significant difference in others,which may be due to large differences among individuals. The level of cytokines (including IFN-gama,IL-4 and IL-12 p70) and NK cell subtype marker in two types of Gal antigen-deficient mice after RRBC administration made no significant differences. Conclusion:Two types of Gal antigen-deficient mice both showed strong sensitivity to foreign antigen stimulation,with a significant increase in the level of specific anti-Gal antibody. GGTA1/iGb3S DKO mice did not show a higher sensitivity than the GGTA1 KO mice. Therefore,as an immunological evaluation model for heterogeneous immunogenic assessment,both GGTA1 KO mice and GGTA1/iGb3S DKO mice are the sensitive experimental animal models. Rabbit red blood cells pre-immunization was applied to improve anti-Gal antibody level in two types of Gal antigen-deficient mice without causing significant immunotoxicity. The research lays a foundation for the later application of experimental animal models for immunological evaluation of animal derived biomaterials.