目的:对于短肠综合征患者,肠道适应是实现肠内独立所必需的。虽然适应已经在动物模型中进行了广泛的研究,对人体肠道的这一过程知之甚少。我们假设对有和没有管腔流动的匹配标本的分析可以识别新的潜在治疗途径。
方法:在短节段肠切除和转移后的回肠造口术逆转手术期间,从2-20个月的儿童中收集了15个配对的人回肠样本。暴露于肠内喂养的节段被表示为喂养,转移的部分被标记为未进料。组织学上比较了形态计量学和细胞分化。RNA测序和基因本体论富集分析鉴定了过度表达和表达不足的途径。免疫荧光染色和Western印迹评估了感兴趣的蛋白质。将配对数据与1尾Wilcoxon秩和检验进行比较,P值小于0.05,被认为是显着的。
结果:未喂养的回肠含有较短的绒毛,较浅的隐窝,和更少的潘氏细胞。缺乏机械腔刺激而上调的基因参与消化,新陈代谢,和运输。LGR5的信使RNA表达在未进食的肠道中明显更高,伴随着磷酸化信号转导和转录激活因子3蛋白水平的增加,和CCND1和C-MYC信使RNA。然而,增殖减少,LGR5+减少,OLFM4+,在未进食的回肠中观察到SOX9肠干细胞(ISC)。
结论:即使有足够的全身热量摄入,人类回肠通过上调刷状边界酶对慢性缺乏机械腔刺激的反应,运输商,结构基因,和ISC基因LGR5和ASCL2。这些数据表明,在重新引入肠内喂养后,未喂养的肠道已准备好补充ISC种群。因此,阐明参与这些过程的通路可能为肠衰竭患者提供治疗靶点.RNA测序数据可从基因表达综合系列GSE82147获得。
OBJECTIVE: For patients with short-bowel syndrome, intestinal adaptation is required to achieve enteral independence. Although adaptation has been studied extensively in animal models, little is known about this process in human intestine. We hypothesized that analysis of matched specimens with and without luminal flow could identify new potential therapeutic pathways.
METHODS: Fifteen paired human ileum samples were collected from children aged 2-20 months during ileostomy-reversal surgery after short-segment intestinal resection and diversion. The segment exposed to enteral feeding was denoted as fed, and the diverted segment was labeled as unfed. Morphometrics and cell differentiation were compared histologically. RNA Sequencing and Gene Ontology Enrichment Analysis identified over-represented and under-represented pathways. Immunofluorescence staining and Western blot evaluated proteins of interest. Paired data were compared with 1-tailed Wilcoxon rank-sum tests with a P value less than .05 considered significant.
RESULTS: Unfed ileum contained shorter villi, shallower crypts, and fewer Paneth cells. Genes up-regulated by the absence of mechanoluminal stimulation were involved in digestion, metabolism, and transport. Messenger RNA expression of LGR5 was significantly higher in unfed intestine, accompanied by increased levels of phosphorylated signal transducer and activator of transcription 3 protein, and CCND1 and C-MYC messenger RNA. However, decreased proliferation and fewer LGR5+, OLFM4+, and SOX9+ intestinal stem cells (ISCs) were observed in unfed ileum.
CONCLUSIONS: Even with sufficient systemic caloric intake, human ileum responds to the chronic absence of mechanoluminal stimulation by up-regulating brush-border enzymes, transporters, structural genes, and ISC genes LGR5 and ASCL2. These data suggest that unfed intestine is primed to replenish the ISC population upon re-introduction of enteral feeding. Therefore, the elucidation of pathways involved in these processes may provide therapeutic targets for patients with intestinal failure. RNA sequencing data are available at Gene Expression Omnibus series GSE82147.