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| 采用改良后的体内哺乳动物肝细胞程序外DNA合成试验检测两种组织工程材料的遗传毒性 |
| Application of a Modified In Vivo Mammalian Hepatocyte Unscheduled DNA Synthesis Test in the Genotoxicity Assessment of Tissue Engineered Materials |
| 投稿时间：2025-04-29 修订日期：2025-04-29 |
| DOI： |
| 中文关键词: 遗传毒性 程序外DNA合成试验 5-乙炔基-2''-脱氧尿苷 组织工程材料 |
| 英文关键词: Genotoxicity Unscheduled DNA Synthesis 5-ethynyl-2-deoxyuridine Tissue Engineered Materials |
| 基金项目:国家重点研发计划(2022YFC2409805) |
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| 摘要点击次数: 6 |
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| 中文摘要: |
| 目的：采用体内哺乳动物肝细胞程序外DNA合成（UDS）试验，检测医用交联透明质酸钠凝胶和骨修复材料的遗传毒性，为医疗器械及其材料的遗传毒性体内UDS试验方法的建立提供依据。方法：采用0.9 %氯化钠注射液和棉籽油两种介质制备试验液，以0.9 %氯化钠注射液和棉籽油作为阴性对照，早期采样组N-二甲基亚硝胺（DMN）作为阳性对照，晚期采样组2-乙酰氨基芴（2-AFF）作为阳性对照。选取SD大鼠，单次给药染毒后分别于4 h和16 h取材，通过胶原酶灌注法制备肝细胞。肝细胞经5-乙炔基-2''-脱氧尿苷（EdU）标记后通过荧光显微镜观察细胞核与核质的净平均荧光强度。为保证检测的准确性与科学性以50个细胞的净平均荧光强度作为分析指标，以此判断DNA修复程度。结果：与阴性对照组相比，DMN 和 2-AFF 阳性对照组的净平均荧光强度差异均具有高度统计学意义（P<0.01），表明阳性对照成功引发 DNA 损伤。而医用交联透明质酸钠凝胶和骨修复材料组间的差异无统计学意义（P>0.05），说明在本实验条件下，两种材料未显著诱导 DNA 损伤。结论：本研究首次将 EdU 标记技术运用到肝细胞 UDS 试验中，该方法不具有放射性，通过与叠氮化合物发生点击反应进行标记，极大提升了检测的便捷性与高效性。在本研究条件下，医用交联透明质酸钠凝胶和骨修复材料的试验液不能诱导大鼠肝细胞 DNA 损伤，未检测出遗传毒性。后续可扩大样本量、延长观察周期，进一步探究不同条件下医疗器械的遗传毒性，为医疗器械的安全性评价提供更全面的数据支持。 |
| 英文摘要: |
| Objective: An in vivo mammalian hepatocyte Unscheduled DNA Synthesis (UDS) test was used to detect genotoxicity of Medical Cross-linked Sodium Hyaluronate Gel and Bone Repair Material, and to develop the method for genotoxicity evaluation of medical devices. Methods: Test solutions were prepared using two media: 0.9% sodium chloride injection and cottonseed oil. 0.9% sodium chloride injection and cottonseed oil served as negative controls. N-dimethylnitrosamine (DMN) was used as the positive control for the early sampling times, and 2-acetylaminofluorene (2-AAF) was used as the positive control for the late sampling times. SD rats were selected, and hepatocytes were prepared by collagenase perfusion after single-dose administration and sampling at 4 hours and 16 hours. Hepatocytes were labeled with 5-ethynyl-2''-deoxyuridine (EdU), and the net average fluorescence intensity (NAFI) of cell nuclei and nucleoplasm was observed using a fluorescence microscope. To ensure accuracy and scientific validity, the NAFI of 50 cells was used as the analytical index to evaluate DNA repair levels. Results: Compared with the negative control groups, the positive control groups (DMN and 2-AAF) showed highly statistically significant differences in NAFI (P<0.01), indicating that positive controls successfully induced DNA damage. In contrast, no statistically significant differences were observed between the medical cross-linked sodium hyaluronate gel and bone repair material groups (P>0.05), suggesting that these materials did not significantly induce DNA damage under the experimental conditions. Conclusion: This study first applied EdU labeling technology to the hepatic UDS assay. This non-radioactive method uses click reactions with azide compounds for labeling, greatly improving the convenience and efficiency of detection. Under the conditions of this study, the test solutions of medical cross-linked sodium hyaluronate gel and bone repair materials did not induce DNA damage in rat hepatocytes, and no genotoxicity was detected. Future research could expand the sample size and observation period to further investigate the genotoxicity of medical devices under different conditions, providing more comprehensive data support for the safety evaluation of medical devices. |
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