皮肤致敏性替代试验方法的研究进展

doi:10.12300/j.issn.1674-5817.2021.173

摘要/Abstract

摘要：

过敏性接触性皮炎是皮肤反复接触某种物质后所引起的Ⅳ型超敏反应，也是常见的公共健康问题。传统皮肤致敏性测试以豚鼠最大值试验、封闭斑贴等动物实验为主。近年来，随着动物伦理不断被人们重视以及科学技术的发展，皮肤致敏试验替代方法相继出现。根据原理不同，这些替代方法分为体内替代法、几类基于有害结局通路的体外替代法、基因组过敏原快速检测法等。本文将对这些皮肤致敏试验替代方法的研究进展以及几类基于有害结局通路的整合测试与评估方法进行综述。

关键词: 皮肤致敏性试验, 过敏性接触性皮炎, 替代方法

Abstract:

Allergic contact dermatitis is a type Ⅳ hypersensitivity reaction caused by repeated skin exposure to a substance and is a common public health problem. Traditional skin sensitization tests are based on animal experiments such as guinea pig maximum test and closed patch. In recent years, with the increasing attention of animal ethics and the development of science and technology, alternative methods of skin sensitization test have emerged. According to different principles, these alternative methods are divided into in vivo alternative methods, several in vitro alternative methods based on harmful outcome pathways, and genomic allergen rapid test, etc. In this paper, we reviewed the progress of these alternative methods of skin sensitization test, and several integrated testing and evaluation methods based on adverse outcome pathways.

Key words: Skin sensitization experiment, Allergic contact dermatitis, Alternative method

中图分类号:

R-332

黄静怡, 李培宁, 刘香梅, 刘忠华, 黄宇锋. 皮肤致敏性替代试验方法的研究进展[J]. 实验动物与比较医学, 2022, 42(4): 313-321.

Jingyi HUANG, Peining LI, Xiangmei LIU, Zhonghua LIU, Yufeng HUANG. Research Progress on Alternative Methods of Skin Sensitization Test[J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 313-321.

图/表 1

参考文献 68

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方法 Method	适用范围 Application scope	优点 Advantages	局限性 Limitation	检测终点 Detection endpoint	准确度 Accuracy
LLNA	化学品 (包括化妆品、农药等)、脂溶性小分子物质	减少实验时间和成本，动物用量少，体积小，灵敏度高	非致敏的皮肤刺激性化学物质^[40]；溶剂假阳性结果^[41]；对受试物溶解性要求高；需要使用实验动物、放射性物质	强致敏剂：SI≥3，EC3<0.1%	72%^[42]
LLNA: BrdU-ELISA	化学品 (包括化妆品、农药等)	综合了LLNA的优点，且未使用放射性物质	未能解决LLNA法中出现的假阳性问题^[43]	ELISA检测增殖淋巴细胞中BrdU的标记数	86%^[44]
LLNA: DA	化学品 (包括化妆品、农药等)	具有LLNA：BrdU-ELISA的全部优点	时间限制；假阳性；不适用于影响ATP产生的化合物	ATP数量、淋巴结质量、增殖的淋巴细胞数，以及SI和EC值	87%^[45]
DPRA	可溶性的单一化学物质以及化妆品原料	不使用实验动物，灵敏度和准确性高	缺乏代谢体系^[46]；溶解性、透皮性差的待测物会影响结果^[47]；待测物与亲核试剂共同洗脱导致准确性下降	液相色谱法测定被测化合物消耗半胱氨酸和赖氨酸多肽的情况	80%
ADRA	可溶性的单一化学物质以及化妆品原料	不使用实验动物，灵敏度和准确性高，对待测物浓度要求低	对待测物溶解性要求较高；评估多成分物质时无法避免待测物的免疫抑制	NAC/NAL的反应性	86.9%^[48]
KeratinoSens^TM/ LuSens	可溶并能形成稳定体系的待测物	灵敏度高，准确度高，不使用实验动物	选择性与赖氨酸残基反应的待测物会出现假阳性^[49]；如待测物需要酶的激活可能出现假阳性^[50]	荧光诱导倍数FI>1.5可判断受试物为致敏物	94%/83%
h-CLAT	可溶性化合物	不使用实验动物，细胞株易于获得且稳定，灵敏度高，特异性高	对待测物的溶解度要求较高；荧光干扰以及待测物细胞毒性引起的假阳性^[51]；弱致敏物质易产生假阴性结果^[52]	当CD86>150%且 CD54>200%时，可判定为致敏物	84%
GARD	可溶性固/液化合物	准确度高，不使用实验动物，稳定、高重复性，可检测呼吸道致敏物	成本高；不能涵盖完整的AOP通路	DC>0，为致敏剂；DC≤0为非致敏剂	84%^[53]

方法 Method	适用范围 Application scope	优点 Advantages	局限性 Limitation	检测终点 Detection endpoint	准确度 Accuracy
LLNA	化学品 (包括化妆品、农药等)、脂溶性小分子物质	减少实验时间和成本，动物用量少，体积小，灵敏度高	非致敏的皮肤刺激性化学物质^[40]；溶剂假阳性结果^[41]；对受试物溶解性要求高；需要使用实验动物、放射性物质	强致敏剂：SI≥3，EC3<0.1%	72%^[42]
LLNA: BrdU-ELISA	化学品 (包括化妆品、农药等)	综合了LLNA的优点，且未使用放射性物质	未能解决LLNA法中出现的假阳性问题^[43]	ELISA检测增殖淋巴细胞中BrdU的标记数	86%^[44]
LLNA: DA	化学品 (包括化妆品、农药等)	具有LLNA：BrdU-ELISA的全部优点	时间限制；假阳性；不适用于影响ATP产生的化合物	ATP数量、淋巴结质量、增殖的淋巴细胞数，以及SI和EC值	87%^[45]
DPRA	可溶性的单一化学物质以及化妆品原料	不使用实验动物，灵敏度和准确性高	缺乏代谢体系^[46]；溶解性、透皮性差的待测物会影响结果^[47]；待测物与亲核试剂共同洗脱导致准确性下降	液相色谱法测定被测化合物消耗半胱氨酸和赖氨酸多肽的情况	80%
ADRA	可溶性的单一化学物质以及化妆品原料	不使用实验动物，灵敏度和准确性高，对待测物浓度要求低	对待测物溶解性要求较高；评估多成分物质时无法避免待测物的免疫抑制	NAC/NAL的反应性	86.9%^[48]
KeratinoSens^TM/ LuSens	可溶并能形成稳定体系的待测物	灵敏度高，准确度高，不使用实验动物	选择性与赖氨酸残基反应的待测物会出现假阳性^[49]；如待测物需要酶的激活可能出现假阳性^[50]	荧光诱导倍数FI>1.5可判断受试物为致敏物	94%/83%
h-CLAT	可溶性化合物	不使用实验动物，细胞株易于获得且稳定，灵敏度高，特异性高	对待测物的溶解度要求较高；荧光干扰以及待测物细胞毒性引起的假阳性^[51]；弱致敏物质易产生假阴性结果^[52]	当CD86>150%且 CD54>200%时，可判定为致敏物	84%
GARD	可溶性固/液化合物	准确度高，不使用实验动物，稳定、高重复性，可检测呼吸道致敏物	成本高；不能涵盖完整的AOP通路	DC>0，为致敏剂；DC≤0为非致敏剂	84%^[53]

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