PDF(Pubmed)
Abstract：
Bacterial overgrowth is common for improperly stored urine. However, its effects on human urinary extracellular vesicles (uEVs) study had not been previously examined nor documented. This study investigated the presence of bacterial EVs (bEVs) contaminated in uEVs samples and their effects on uEVs study. Nanoscale uEVs were isolated from normal human urine immediately after collection (0-h) or after 25°C-storage with/without preservative (10 mM NaN3) for up to 24-h. Turbidity, bacterial count and total uEVs proteins abnormally increased in the 8-h and 24-h-stored urine without NaN3. NanoLC-ESI-LTQ-Orbitrap MS/MS identified 6-13 bacterial proteins in these contaminated uEVs samples. PCR also detected bacterial DNAs in these contaminated uEVs samples. Besides, uEVs derived from 8-h and 24-h urine without NaN3 induced macrophage activation (CD11b and phagocytosis) and secretion of cytokines (IFN-α, IL-8, and TGF-β) from macrophages and renal cells (HEK-293, HK-2, and MDCK). All of these effects induced by bacterial contamination were partially/completely prevented by NaN3. Interestingly, macrophage activation and cytokine secretion were also induced by bEVs purified from Escherichia coli. This study clearly shows evidence of bEVs contamination and their effects on human uEVs study when the urine samples were inappropriately stored, whereas NaN3 can partially/completely prevent such effects from the contaminated bEVs.
摘要：
细菌过度生长对于储存不当的尿液是常见的。然而,其对人尿细胞外囊泡(uEVs)研究的影响以前没有检查,也没有记录.这项研究调查了uEV样品中细菌EV（bEV）的存在及其对uEV研究的影响。收集后（0小时）或在25°C下储存/不含防腐剂（10mMNaN3）长达24小时后，立即从正常人尿液中分离出纳米级uEV。浊度,在没有NaN3的情况下，8小时和24小时储存的尿液中细菌计数和总uEVs蛋白异常增加。NanoLC-ESI-LTQ-OrbitrapMS/MS在这些受污染的uEV样品中鉴定出6-13种细菌蛋白。PCR还在这些受污染的uEV样品中检测到细菌DNA。此外,来自8小时和24小时尿液的uEV，没有NaN3诱导的巨噬细胞活化（CD11b和吞噬作用）和细胞因子的分泌（IFN-α，IL-8和TGF-β）来自巨噬细胞和肾细胞（HEK-293，HK-2和MDCK）。NaN3部分/完全阻止了由细菌污染引起的所有这些作用。有趣的是,从大肠杆菌纯化的bEV也诱导巨噬细胞活化和细胞因子分泌。这项研究清楚地表明，当尿液样本被不适当地储存时，bEV污染的证据及其对人类uEV研究的影响，而NaN3可以部分/完全防止受污染的bEV的这种影响。
