Genome-wide analysis of gene expression in jnk1-/-jnk2-/- immortalized mouse embryonic fibroblasts compared to wildtype counterparts co-cultivated with differentiating keratinocytes. Mus musculus

Analysis of JNK-dependent fibroblast-derived soluble factors at gene expression level. The hypothesis tested in the present study was that loss of c-Jun N-terminal kinases 1 and 2, JNK1 and JNK2, in MEFs causes a strong alteration of the gene expression program coding for soluble factors, which promote an efficient keratinocyte differentiation. Results provide important information of the repertoire of fibroblast transcripts encoding secreted proteins, which is severely disarranged upon loss of JNK activity under the in vitro co-culture conditions applied. Overall design: In vitro transwell co-culture experiments were performed using jnk1-/-jnk2-/- or wildtype immortalized mouse embryonic fibroblasts (MEFs) and differentiating primary normal human epidermal keratinocytes (NHEK) over a time course of 6 days. Every second day, fibroblast-loaded inserts were changed resulting in 3 triads (triads 1, 2, and 3). Total RNA was obtained from jnk1-/-jnk2-/- and wildtype immortalized mouse embryonic fibroblasts (MEFs) prior to co-cultivation (day 0) and of each triad 1, 2, or 3.

本研究针对JNK依赖的成纤维细胞来源可溶性因子开展基因表达水平分析。本实验所检验的假说为：永生化小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）中c-Jun氨基末端激酶（c-Jun N-terminal kinases, JNK）1和2（JNK1、JNK2）的缺失，会显著改变编码可溶性因子的基因表达程序，进而有效促进角质形成细胞分化。研究结果提供了成纤维细胞分泌蛋白编码转录本完整表达谱的关键信息，在本研究所采用的体外共培养条件下，该转录谱因JNK活性缺失发生严重紊乱。
整体实验设计：采用jnk1-/-jnk2-/-敲除型与野生型永生化小鼠胚胎成纤维细胞（MEFs），与正在分化的原代正常人表皮角质形成细胞（normal human epidermal keratinocytes, NHEK）进行体外Transwell共培养实验，培养周期为6天。期间每2天更换一次接种有成纤维细胞的Transwell小室，共设置3个生物学重复组（重复组1、2、3）。在共培养起始前（第0天）以及每个重复组的对应节点，分别收集jnk1-/-jnk2-/-敲除型与野生型永生化小鼠胚胎成纤维细胞的总RNA。