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Received:May 14, 2018 Published Online:December 21, 2018
Received:May 14, 2018 Published Online:December 21, 2018
中文摘要: 目的 通过检测炎症性肠病(IBD)患者肠外泌体(EVs)对上皮细胞和巨噬细胞的影响,研究EVs对IBD的影响。 方法 选择2016年1月至2017年12月收治的40例IBD患者及肠镜未见异常,既往无慢性病史的正常人群20例作为健康对照组(HC组),每例入选者行肠镜检查时收集10 ml肠腔液(LAs),分离EVs。将EVs与结肠上皮细胞株(SW-480细胞系细胞)或者外周血单核细胞来源的巨噬细胞共培养,分别采用qRT-PCR及ELISA法检测LAs中EVs裂解液中细胞因子mRNA表达水平及其蛋白含量,及与EVs共培养的结肠上皮细胞及巨噬细胞吞噬肠EVs后细胞因子mRNA表达水平和蛋白水平,并行巨噬细胞迁移实验探讨EVs对巨噬细胞功能的影响。 结果 (1)与健康受试者(HC)比较,IBD患者LAs中EVs(IBD-EVs)裂解液中白介素(IL)-6、IL-8、IL-10、肿瘤坏死因子-α(TNF-α)的mRNA及蛋白表达水平均显著升高,转化生长因子β(TGF-β)及黏蛋白-2(MUC-2)mRNA及蛋白表达水平均显著降低。(2)与 IBD-EVs共培养的结肠上皮细胞吞噬EVs后IL-6、IL-8、TNF-α的mRNA表达水平及其蛋白水平均显著高于与HC-EVs共培养的水平,而TGF-β及MUC-2 mRNA及蛋白表达水平均显著低于与 HC-EVs共培养时的水平,巨噬细胞吞噬肠腔EVs后细胞因子水平变化与上皮细胞类似,且IBD患者EVs显著提升巨噬细胞的迁移率。 结论 IBD患者肠腔中释放的EVs的内含物与健康受试者不同,体外实验表明 IBD-EVs具有促进肠道上皮细胞、巨噬细胞发生炎症的作用,而该类小体的稳定性及其包含的mRNA及蛋白谱等特性可能作为临床诊断IBD时潜在的标志物。
Abstract:Objective To investigate the effect of extracellular vesicles (EVs) on inflammatory bowel disease. (IBD) via detecting the influence of intestinal luminal EVs on intestinal epithelial cells and macrophages.? Methods Forty patients with IBD from January 2016 to December 2017 and 20 normal population without abnormalities in enteroscopy and without chronic diseases history (HC group) were selected as research objective. The 10 ml intestinal luminal aspirates (LAs) were collected during colonoscopy in all chosen subjects, and the EVs were isolated. EVs were co-cultured with colon epithelial cell line SW-480 cells or macrophages derived from peripheral blood monocytes in vitro. Real-time quantitative polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect cytokines mRNAs expressions and contents of their proteins in lysis buffer of EVs and in epithelial cells and macrophages after swallow EVs Macrophage migration test was performed to explore the effect of EVs on functions of macrophages.? Results (1) Compared with healthy subjects (HC), the expression levels of interleukin (IL) -6, IL-8, IL-10, tumor necrosis factor-α (TNF-α) mRNAs and their proteins contents in lysis buffer of EVs of IBD patients LAs (IBD-EVs) increased significantly, and the expression levels of transforming growth factor-β (TGF-β) and mucoprotein-2 (MUC-2) mRNAs and their protein contents decreased significantly. (2) Compared with co-culture with HC-EVs, the expression levels of IL-6, IL-8, IL-10, TNF-α mRNAs and their proteins contents in epithelial cells co-cultured with IBD-EVs after swallow EVs increased significantly, and the expression levels of TGF-β and MUC-2 mRNAs and their protein contents decreased significantly. The changes of cytokine levels in macrophages co-cultured with IBD-EVs after swallow EVs were similar to epithelial cells co-cultured with IBD-EVs after swallow EVs, and IBD-EVs can significantly increase the migration rate of macrophage.? Conclusion The inclusions released by EVs in LAs of IBD patients are different from HC. The experiment in vitro shows that IBD-EVs have the functions of promoting inflammation of intestinal epithelial cells and macrophages, and their stability and the characteristics of mRNA and protein profiles contained in them may be used as the potential fecal markers for clinical diagnosis of IBD.
文章编号: 中图分类号:R 574 文献标志码:B
基金项目:江苏省自然科学基金青年基金项目(BK20170372);南京医科大学科技发展基金重点项目(2017NJMUZD110)
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