Immuno-genomic effects of JAK blockade in vivo. Mus musculus

Small molecule inhibitors of JAK kinases have shown clinical effcacy in the treatment of certain autoimmune diseases. While these are known to block upstream JAK signalling events, their broader impact on the transcriptional footprint in immunocytes are unknown. Here we explore the effects of pan- and isoform-specific JAK blockade on the immuno-genomic network by genomic profiling. Overall design: 6week old male C57BL/6 mice were gavaged with JAK inhibitor or vehicle for indicated treatment periods. Spleens were harvested and mechanically disrupted to prepare single cell suspensions. These were then stained in multiple surface marker panels to differentiate distinct immunocyte populations. Cells were sorted directly into TriZol. RNA was prepared in Trizol for gene expression profiling by Affymetrix Mouse Gene 1.0 ST Arrays.JAK激酶 (Janus Kinase) 的小分子抑制剂在部分自身免疫性疾病的治疗中已展现出临床疗效。尽管已知此类抑制剂可阻断上游JAK信号通路事件，但它们对免疫细胞中转录足迹 (transcriptional footprint) 的整体影响仍不明晰。本研究通过基因组谱分析 (genomic profiling)，探究泛JAK及亚型特异性JAK阻断对免疫基因组网络的调控效应。 总体实验设计：选取6周龄雄性C57BL/6小鼠，经口灌胃给予JAK抑制剂或赋形剂 (vehicle)，处理时长遵循实验设定要求。采集小鼠脾脏并通过机械解离制备单细胞悬液；随后使用多组表面标志物染色组合对细胞进行染色，以区分不同免疫细胞群。将细胞直接分选收集至TriZol试剂中，采用TriZol法提取RNA，通过Affymetrix小鼠基因1.0 ST芯片阵列开展基因表达谱分析。