Expression data from human bronchial epithelial cells. Homo sapiens

The F508del mutation, the most frequent in cystic fibrosis (CF), impairs the maturation of the CFTR chloride channel. The F508del defect can be partially overcome at low temperature (27 °C) or with pharmacological correctors. The rescue elicited by low temperature may involve a direct stabilization of mutant CFTR protein and/or a change in cell transcriptome that creates a more favorable proteostasis environment. To assess the effect of low temperature on gene expression we investigated the transcriptome of bronchial epithelial cells derived from CF with F508del mutation. Cells were kept under control conditions or incubated at 27 °C. Microarray data indicate that hypothermia induces a profound and global change in gene expression that may be in part responsible for rescue of F508del-CFTR. Overall design: Bronchial epithelial cells from cystic fibrosis patients homozygotes for F508del mutation were isolated. Cells differentiated as epithelial monolayers on porous membranes (Snapwell inserts) were incubated for 24 hours at 27 °C or kept under control conditions. Rescue of mutant CFTR channel by low temperature was checked by measuring transepithelial chloride currents with the Ussing chamber technique. Total RNA was extracted from treated and control cells to assess changes in gene expression with microarrays.

F508del突变是囊性纤维化（cystic fibrosis, CF）最常见的致病突变，可损害囊性纤维化跨膜传导调节因子（CFTR）氯离子通道的成熟过程。F508del突变导致的功能缺陷可通过低温（27℃）处理或使用药理学矫正剂得到部分挽救。低温诱导的挽救效应可能涉及突变型CFTR蛋白的直接稳定，以及/或改变细胞转录组，从而构建更利于蛋白稳态（proteostasis）的细胞内环境。为评估低温对基因表达的调控作用，本研究对携带F508del突变的囊性纤维化患者来源的支气管上皮细胞的转录组进行了分析。实验中将细胞分为两组：一组维持于常规培养条件（对照组），另一组于27℃下孵育。微阵列（Microarray）检测数据显示，低温诱导了基因表达的广泛且全局性改变，这或许部分参与了F508del-CFTR的功能挽救过程。整体实验设计：从携带F508del纯合突变的囊性纤维化患者体内分离支气管上皮细胞；将细胞在多孔膜（Snapwell插入式培养板）上诱导分化为上皮单层后，分别于27℃下孵育24小时，或维持于常规培养条件；通过尤斯腔室（Ussing chamber）技术检测跨上皮氯离子电流，以验证低温对突变型CFTR通道的挽救效果；提取处理组与对照组细胞的总RNA，利用微阵列芯片分析基因表达的变化情况。