Development of a new, predictive in vitro test for screening of allergic co ntact sensitizers

开发一种新的预测性体外测试，用于筛选过敏性接触致敏剂

• 批准号: 13672349
• 负责人: TABATA Masako
• 金额: $ 2.05万
• 依托单位: Tokyo University of Sciences Faculty of Pharmaceutical Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672349/
• 关键词: contact dermatitis; keratinocvtes; Langerhans cells; cytokine; chemokine; HaCaT; ELD-1; cDNA microarray; ケモカインレセプター

To develop a simple predictive in vitro test for screening of chemical substances that induce allergic contact dermatitis, we examined candidate markers using the HaCaT epidermal keratinocyte cell line, and the ELD-1Langerhans cell line.Expression of cytokine and chemokines mRNA by ELD-1 and HaCaT cells exposed to known allergic contact sensitizers was analyzed quantitatively by real-time reverse transcription-polyinerase chain reaction (RT-PCR).In HaCaT cells, level of mRNA for interlurkin-1α(IL-α), tumor necrosis factor(TNF) and chemokine MIP-3α were increased by allergic contact sensitizers, whereas mRNA levels were altered only slight by ELD-1 cells.When HaCaT and ELD-1 cells were exposed by IL-1α and TNF, which are secreted by keratinocytes, mRNA level and secretion of IL-1α, TNF, and MIP-3α were increased remarkably in HaCaT cells, and IL-6 and IL-8 were detected in cultured media of ELD-1 cells.These findings suggest that keratinocytes and Langerhans cells interact closely during induction of allergic contact dermatitis.To analyze more comprehensively changes in RNA expression in response to allergens, we exposed HaCaT and ELD-1 cells to dinitrochlorobenzene(DNCB), an allergic contact sensitizers, and benzalkonium chloride(BC), a skin irritant, and we then examined changes in mRNA expression change by cDNA microarray method.DNCB increased changes in expression of more genes than did BC in both cell lines. Expression of four genes was increased significantly and expression of two genes was decreased significantly in HaCaT cells.Expression of four genes was decreased significantly in ELD-1 cells.In the present study, we used cutaneous cell lines to investigate expression of several candidate genes as indices of allergic sensitization.We plan to investigate marker molecules in a keratinocyte-Langerhans cell co-culture system.

为了开发一种用于筛选诱导变应性接触性皮炎的化学物质的简单预测性体外试验，我们使用HaCaT表皮角质形成细胞系检查了候选标志物，通过实时逆转录-聚合酶链反应（RT-PCR）定量分析已知过敏性接触致敏剂暴露的ELD-1和HaCaT细胞的细胞因子和趋化因子mRNA表达。在HaCaT细胞中，IL-1α、TNF和MIP-3α的mRNA水平被过敏性接触致敏剂所增加，而ELD-1细胞仅轻微改变其mRNA水平，当HaCaT和ELD-1细胞暴露于角质形成细胞分泌的IL-1 α和TNF时，结果提示，在变应性接触性皮炎的发生过程中，角质形成细胞和朗格汉斯细胞之间存在着密切的相互作用。我们将HaCaT和ELD-1细胞暴露于过敏性接触致敏剂二硝基氯苯（DNCB）和皮肤刺激剂苯扎氯铵（BC），然后通过cDNA微阵列方法检测mRNA表达的变化。HaCaT细胞中有4个基因表达显著增加，2个基因表达显著降低，ELD-1细胞中有4个基因表达显著降低。在本研究中，我们使用皮肤细胞系来研究几个候选基因的表达作为过敏性致敏的指标。我们计划在角质形成细胞-朗格汉斯细胞共培养系统中研究标记分子。