Gene expression changes in the microdissected colon epithelium are similar to those of intact colon in the Il10-/- mouse model of IBD

Epithelial cells play an important role in the protection of the colon mucosa from the resident microbiota and are involved in the initiation and maintenance of intestinal inflammation. LMD is a technique that allows the extraction of specific cell types, such as colonic epithelial cells, to analyse gene expression. LMD of colon epithelial cells followed by microarray analysis could be of more value than microarray analysis of intact colon for determining which pathways are active in the colon mucosa in the early and late stages of inflammation due to increased sensitivity to changes in specific cell populations. An experiment was performed using microarray analysis of intact colon samples and microdissected colon epithelial cell samples from Il10-/- and C57BL/6J mice at 6 and 12 weeks of age to study the molecular changes that occur in early and late inflammation stages in colon epithelium of a mouse model of colitis. Results showed that intact colon and colon epithelial cell gene expression profiles were similar in terms of pathways between Il10-/- and C57BL/6J mice at 12 weeks of age and between Il10-/- mice at 12 and 6 weeks of age. More immune-related pathways were identified at 6 weeks of age in epithelial cells than intact colon. This suggests that LMD and targeting of specific cell types may be of particular use when studying the early stages of inflammation before the intestinal morphology is detectably altered, by increasing analysis sensitivity to mucosal gene expression changes. 2x2 factorial with two tissue types analysed. Two strains of mouse (Il10 knockout mouse and the background strain C57BL/6J) were sampled at 2 timepoints (6 and 12 weeks of age) and intact proximal colon and colon epithelium harvested from each mouse (6 mice per group except for group colon epithelium C57 mouse 12 weeks where only 5 samples reached quality control standards).上皮细胞在保护结肠黏膜免受常驻菌群侵害方面发挥关键作用，并参与肠道炎症的启动与维持。激光显微切割（Laser Microdissection, LMD）是一种可精准提取特定细胞类型（如结肠上皮细胞）以开展基因表达分析的技术。对结肠上皮细胞实施LMD后再进行微阵列分析（microarray analysis），相较于完整结肠组织的微阵列分析，在鉴定炎症早晚期结肠黏膜中活跃的通路时更具应用价值，因其对特定细胞群体的变化具有更高的检测灵敏度。 本实验以结肠炎小鼠模型为研究对象，分别于6周龄和12周龄时采集Il10基因敲除（Il10-/-）小鼠与背景品系C57BL/6J小鼠的样本，采用完整结肠组织与显微切割获取的结肠上皮细胞样本进行微阵列分析，旨在探究结肠炎小鼠结肠上皮组织在炎症早晚期发生的分子改变。 实验结果显示：在12周龄的Il10-/-与C57BL/6J小鼠之间，以及12周龄与6周龄的Il10-/-小鼠之间，完整结肠组织与结肠上皮细胞的基因表达通路谱均较为相似。相较于完整结肠组织，上皮细胞样本在6周龄时可鉴定出更多免疫相关通路。 上述结果表明，当在肠道形态出现可检测到的改变之前研究炎症早期阶段时，通过提升对黏膜基因表达变化的分析灵敏度，靶向特定细胞类型的LMD技术或可发挥独特的应用价值。本实验采用2×2析因实验设计，对两种组织类型进行分析。实验选取两种小鼠品系（Il10基因敲除小鼠与背景品系C57BL/6J小鼠），在两个时间点（6周龄与12周龄）进行采样，每只小鼠均采集完整近端结肠组织与结肠上皮细胞样本：每组原计划各6只小鼠，仅12周龄C57BL/6J小鼠上皮细胞组仅有5份样本符合质量控制标准。