Induction of inflammatory cytokines by a keratin mutation in a mouse model for EBS. Mus musculus

Epidermolysis bullosa simplex (EBS) is a skin disorder caused by mutations in keratin (K) 5 or K14 genes. It is widely regarded as a mechanobullous disease, resulting from a weakened cytoskeleton, causing extensive cytolysis. It was postulated by others that certain K14 mutations induce TNF-alfa and increase apoptosis. Here, we report that in K5-/- mice , the mRNA and protein levels of TNF-alfa remain unaltered. Transcriptome analysis of K5-/- mice revealed however, that the pro-inflammatory cytokines interleukin-6 and interleukin-1beta were significantly upregulated at the mRNA level in K5-/- mouse skin. These results were confirmed by TaqMan real-time PCR and ELISA assays. We hypothesize that keratin mutations contribute to EBS in a mouse model by inducing local inflammation that mediates a stress response. Keywords: comparative gene expression profile Overall design: Two groups were K5-/- skin and wildtype . Six animals were included in each group. All the total RNA samples were isolated from tissues taken immediately after birth, and were pooled for later microarray experiments. Using realtime PCR and ELISA analysis confirmed our microarray result.

单纯大疱性表皮松解症（Epidermolysis bullosa simplex, EBS）是一类由角蛋白（keratin, K）5或K14基因突变引发的皮肤疾病。该疾病被广泛归类为机械性大疱病，其病理机制为细胞骨架结构脆弱，进而导致广泛的细胞溶解。此前已有研究提出，部分K14突变可诱导肿瘤坏死因子-α（TNF-α）表达并增加细胞凋亡。本研究发现，在K5基因敲除（K5-/-）小鼠中，TNF-α的mRNA与蛋白水平均未发生显著改变。然而，对K5-/-小鼠的转录组分析显示，其皮肤组织中促炎细胞因子白细胞介素-6（interleukin-6, IL-6）与白细胞介素-1β（interleukin-1β, IL-1β）的mRNA水平显著上调。上述结果通过TaqMan实时荧光定量PCR与酶联免疫吸附测定（ELISA）得到了验证。我们提出假说：在该小鼠模型中，角蛋白突变通过诱导局部炎症反应介导应激应答，从而参与单纯大疱性表皮松解症的发病过程。
关键词：比较基因表达谱
整体实验设计：实验分为两组，即K5基因敲除皮肤组与野生型组，每组各纳入6只实验动物。所有总RNA样本均采集自新生即刻的小鼠组织，混合后用于后续的基因芯片实验。本研究通过实时定量PCR与ELISA分析验证了基因芯片的实验结果。