应用Gal抗原缺失小鼠评价可降解异种脱细胞真皮基质的免疫原性

目的：采用Gal抗原缺失小鼠评价脱细胞真皮基质及其降解过程中的残余免疫风险。方法：Gal抗原缺失小鼠经兔血预免后，随机分为对照组、T1组、T2组。T1组腿部肌肉间植入牛真皮基质（原材料），T2组肌肉间植入脱细胞真皮基质，对照组进行"假手术"操作。在植入后的4、12、52周分别取材，用ELISA方法检测小鼠血清总IgG、IgM含量和血清抗-Gal IgG、IgM水平；用流式细胞仪检测血清IL-4、IL-10、IL-12p40、IFN-γ等细胞因子含量，以及脾淋巴细胞亚型百分比等；进行脾体外淋巴细胞增殖试验，采用CCK-8试剂检测淋巴细胞增殖率；取胸腺、脾脏以及包裹植入物的局部肌肉样本，HE染色后进行组织病理学评价。通过与对照组的比较，进行牛真皮基质原材料和脱细胞真皮基质的免疫毒理学风险评价。结果：与对照组相比，小鼠植入牛真皮基质原材料的T1组小鼠血清抗-Gal IgG含量和体外脾脏淋巴细胞增殖率有统计学显著性差异。T1组小鼠在4周时，血清抗-Gal IgG平均含量是对照组的2倍；植入12周时，血清抗-Gal IgG平均含量继续升高，达到对照组的近7倍，统计学分析均有显著性差异，表明由降解引起的Gal抗原持续暴露或暴露增加，致使小鼠血清抗-Gal IgG含量升高；在植入52周时，血清抗-Gal IgG平均含量回复到对照组水平。植入12周时，T1组小鼠的脾淋巴细胞体外培养3、7d时，淋巴细胞增殖率分别是对照组的211%和243%。组织病理学分析显示T1组牛真皮基质植入后4周时局部反应为重度刺激，12周时为中度刺激，52周时为轻微刺激。植入脱细胞真皮基质的T2组在4、12、52周的不同时点，小鼠血清总IgG、IgM含量，抗-Gal IgG和IgM含量，血清细胞因子，脾淋巴细胞分型和淋巴细胞体外增殖等不同指标的检测结果与对照组相比均无统计学差异；组织病理学分析显示T2组脱细胞真皮基质植入后4周时局部反应为中度刺激，12周时为轻微刺激，52周时为无刺激。而且，脱细胞真皮基质在4、12、52周时的植入局部反应明显低于真皮基质原材料。结论：牛真皮基质（原材料）及其降解产物具有引起Gal抗原缺失小鼠血清抗-Gal IgG含量升高和脾脏淋巴细胞增殖的免疫原性；而脱细胞真皮基质在植入后降解过程中的免疫反应与对照组相比均无显著性差异。这些结果表明脱细胞处理工艺降低了脱细胞真皮基质的特异性免疫反应。

Objective: To evaluate the residual immune risk of acellular dermis matrix during the biodegradation using Gal antigen-deficient mice. Methods: Gal antigen-deficient mice were randomly divided into the control group, T1 group and T2 group in subsequent to the preimmunization with rabbit blood. The control group underwent a "false operation", while T1 group was implanted with bovine dermis matrix (raw material)and T2 group was implanted with acellular dermis matrix between the leg muscles of mice. At 4 weeks, 12 weeks and 52 weeks after implantation, the total serum IgG and IgM, and serum anti-Gal IgG and IgM levels were measured by ELISA, and serum IL-4, IL-10, IL-12p40, IFN-γ as well as the percentage of spleen lymphocyte subtypes were detected using flow cytometry, and the proliferation test of spleen lymphocytes in vitro was carried out, in which the proliferation rate of lymphocytes was determined by CCK-8 reagent. Thymus, spleen and local muscle contained implant specimens were stained by HE for a histopathological observation. The immunotoxicological risks of bovine dermis matrix in T1 group and acellular dermis matrix in T2 group were evaluated by comparing with the control group. Results: The serum anti-Gal IgG content and the proliferation rate of spleen lymphocytes in T1 group were significantly different from those in the control group. At 4 weeks after implantation, the average content of serum anti-Gal IgG in T1 group was twice as high as that in the control group, and the average content of serum antiGal IgG continued to increase until 12 weeks after implantation, which was nearly 7 times higher than that in the control group. These results indicated that the continuous exposure or enhanced exposure of xeno antigen (like Gal antigen)caused by the degradation of sample resulted in an increase of serum anti-Gal IgG content in Gal antigendeficient mice. At 52 weeks after implantation, the average content of serum anti-Gal IgG in T1 group returned to the level of that in the control group. At 12 weeks of implantation, the proliferation rate of spleen lymphocytes in T1 group that cultured for 3 days and 7 days were 211% and 243% of that in the control group, respectively. Histopathological data showed that there was severe irritation in local tissues at 4 weeks, moderate irritation at 12 weeks and slight irritation at 52 weeks after implantation of bovine dermis matrix in T1 group. In T2 group, serum total IgG and IgM content, anti-Gal IgG and IgM content, serum cytokines, spleen lymphocyte subtypes and the proliferation rate of lymphocytes in vitro were no significant differences compared to the control group at 4 weeks, 12 weeks and 52 weeks after implantation, and histopathological analysis showed that there was moderate irritation at 4 weeks, mild irritation at 12 weeks and non-irritation at 52 weeks after acellular dermis implantation. Furthermore, the inflammation of acellular dermis matrix at 4 weeks, 12 weeks and 52 weeks after implantation were significantly lower than those of dermis matrix raw materials. Conclusion: The bovine dermis matrix (raw material)and its degradation products can increase of serum anti-Gal IgG content and spleen lymphocytes proliferation in Gal antigen-deficient mice, but there is no significant change in immune response in acellular dermis matrix implantation group during post-implantation and material degradation. These results suggest that decellularizing treatment decreases the adverse immune responses.

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