【摘 要】
：
目的 建立基于Real-time PCR方法的人细胞系激发试验(h-CLAT),探讨该方法在化妆品原料皮肤致敏评价中的应用,同时与基于流式细胞术的Draft OECD TG 442E方法进行比较.方法 选用8个已知皮肤致敏特性的参照物(包括对苯二胺、苯乙醛、氯亚铂酸铵、咪唑烷基脲、硫酸镍、硫酸钴、十二烷基硫酸钠和乳酸)处理THP-1细胞24h后,用基于流式细胞术和Real-time PCR检测共刺
【机 构】
：
安利(中国)研发中心,上海,201203
【出 处】
：
2016(第二届)毒性测试替代方法与转化毒理学(国际)学术研讨会暨有害结局路径(AOP)与风险评估培训会议
论文部分内容阅读
目的 建立基于Real-time PCR方法的人细胞系激发试验(h-CLAT),探讨该方法在化妆品原料皮肤致敏评价中的应用,同时与基于流式细胞术的Draft OECD TG 442E方法进行比较.方法 选用8个已知皮肤致敏特性的参照物(包括对苯二胺、苯乙醛、氯亚铂酸铵、咪唑烷基脲、硫酸镍、硫酸钴、十二烷基硫酸钠和乳酸)处理THP-1细胞24h后,用基于流式细胞术和Real-time PCR检测共刺激分子CD86和CD54的蛋白水平或者mRNA水平.通过比较实验组合对照组中两种共刺激分子的诱导表达改变,根据判断标准判定受试物是否具有皮肤致敏性.对于基于流式细胞术的方法,采用Draft OECD TG 442E中的判断标准(当细胞活力≥50％,RFI CD86≥150和/或RFI CD54≥200可判定为阳性)；对于基于Real-time PCR的方法,则根据CD86和CD54的mRNA诱导表达倍数(Relative mRNA expression level,RMEL)制定判断标准. 结果 1.根据Draft OECD TG 442E中的判断标准,基于流式细胞术的方法能够准确将致敏物与非致敏物分开；2.以细胞活力≥50％,RMEL CD86≥ 150和/或RMEL CD54≥150来判定是否为致敏物时,能够准确将致敏物与非致敏物分开；3.在一个实验周期内,相对于Draft OECD TG 442E法,基于Real-time PCR的检测方法能够将检测通量提高一倍,而且对于操作者而言,后者在时间安排上更为灵活,为实验者提供较大便利；流式细胞学检测中有时会受到来自受试物的荧光干扰,而基于mRNA水平检测的方法则不会受到影响.结论 基于Real-time PCR的h-CLAT方法既可以准确区分致敏物和非致敏物,又能够增加检测通量并便于操作,可用于化妆品原料皮肤致敏性的快速初筛.
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