运用胸苷激酶基因突变实验评价动物源性材料的遗传毒性

背景：目前大量动物来源的医疗器械产品,尤其是与人体直接接触的产品,应明确其力学性能、生物降解行为和生物相容性、细胞毒性、遗传毒性、免疫原性等是否符合临床要求。目的：评价动物源性材料的体外遗传毒性,比较非活化和活化、短期和长期胸苷激酶基因突变实验的异同。方法：按照GBT16886-12(1)制备可吸收性硬脑膜补片(马胶原)和人工生物心脏瓣膜(牛心包片)浸提液,以两种浸提液处理L5178Y小鼠淋巴瘤细胞3,24 h后,采用微孔板法行胸苷激酶基因突变实验,计算接种效率、相对悬浮生长、相对存活率、相对总生长及突变频率等指标,并比较短期和长期,非活化和活化处理的结果。结果与结论：两种生物材料浸提液处理小鼠淋巴瘤细胞3 h或24 h,在活化及非活化条件下,胸苷激酶基因突变实验结果均为阴性。表明在体外遗传毒性实验中未发现可吸收性硬脑膜补片和人工生物心脏瓣膜致L5178Y细胞基因突变的作用,无遗传毒性。

Genetictoxicity of animal original biomaterials evaluated by thymidine kinase gene mutation assay

BACKGROUND:At present, a large number of medical device products originating from animals, especially those in direct contact with human body should be known about whether they conform to the clinical requirements, such as their mechanical property, biodegradable behavior, biocompatibility, cytotoxicity, genotoxicity and immunogenicity.OBJECTIVE:To evaluate the in vitro genotoxicity of two kinds of animal original materials, and to compare the differences and similarities of thymidine kinase gene mutation affected by short and long term, as well as non-activation and activation treatments.METHODS:According to GBT16886-12(1), the artificial biological heart valve and absorbable dura mater patch were extracted. They were consisted mainly of bovine pericardium and horse collagen respectively. L5178Y mouse lymphoma cells were treated with the two kinds of leaching liquors for 3 and 24 hours. Then, thymidine kinase gene mutation assay was performed by microplate method with line. Inoculation efficiency, relative suspension growth, relative survival, relative total growth and mutation frequency were calculated. The results of short and long term, as well as non-activation and activation, treatments were compared.RESULTS AND CONCLUSION:After the mouse lymphoma cells were treated with the two kinds of leaching liquors for 3 and 24 hours in non-activation and activation, the results of thymidine kinase gene mutation assays showed negative. These results suggest that the two kinds of animal original materials have no mutagenic effect on L5178Y cells gene in the test of in vitro genotoxicity, which indicates that these two kinds of animal original materials have no genotoxicity and they are safe with current genotoxicity evaluation methods.