Development of an in vitro skin sensitization test using human cell lines: The human Cell Line Activation Test (h-CLAT) I. Optimization of the h-CLAT protocol
Development of an in vitro skin sensitization test using human cell lines: The human Cell Line Activation Test (h-CLAT) I. Optimization of the h-CLAT protocol
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DOI：
10.1016/j.tiv.2005.10.012
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发表时间：
2006-08-01
影响因子：
3.2
通讯作者：
Toyoda, H
中科院分区：
文献类型：
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作者：
Ashikaga, T;Yoshida, Y;Toyoda, H
本研究的目的是优化使用人细胞系THP-1和U-937进行体外皮肤致敏试验的实验条件。至于预培养时间，与评价的其他时间点相比，DNCB处理的THP-1细胞上的CD 86表达在预培养48 h和72 h后趋于更高。接下来，我们研究了化学处理时间的影响，发现DNCB对THP-1细胞上CD 86表达的诱导在24 h后达到平台。当用亚毒性剂量的过敏原处理细胞时，经常观察到CD 86表达的增强。为了确定试验样品的合适剂量，采用MTT法评估试验样品对THP-1和U-937细胞的细胞毒性，并计算每个试验样品的50%抑制浓度（IC 50）。根据细胞毒性试验数据，选择了毒性和无毒范围内的四种浓度（0.1 x、0.5 x、1 x和2 x IC 50）。测试了几种抗体用于染色用过敏原/非过敏原（例如，DNCB。Ni/SLS），并选择用于染色CD 86和CD 54的合适抗体。我们确认所选CD 86和CD 54抗体的工作稀释液适用于我们的方法。还评价了FcR阻断程序的效果。通过FcR阻断程序降低平均荧光强度（MFI值），这表明非特异性染色被阻断。因此，该方法中应包括该程序。根据我们的研究结果，优化了该试验的方案，并确定了未来验证研究中使用的实验条件。我们建议将这种体外皮肤致敏试验称为h-CLAT，是人类细胞系激活试验的缩写。(c)2005爱思唯尔有限公司保留所有权利。
The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48 h and 72 h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24 h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1 x, 0.5x, 1 x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB. Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test. (c) 2005 Elsevier Ltd. All rights reserved.