PDF(Pubmed)
Abstract：
Human milk contains extracellular vesicles (HMEVs). Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aimed to develop an EV isolation pipeline from small volumes of human milk and neonatal intestinal contents after milk feeding (digesta) to address the hypothesis that HMEVs survive in vivo neonatal digestion to be taken up intestinal epithelial cells (IECs). Digesta was collected from nasoduodenal sampling tubes or ostomies. EVs were isolated from raw and pasteurized human milk and digesta by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, western blotting, nanoparticle tracking analysis, resistive pulse sensing, and super-resolution microscopy. EV uptake was tested in human neonatal enteroids. HMEVs and digesta EVs (dEVs) show typical EV morphology and are enriched in CD81 and CD9, but depleted of β-casein and lactalbumin. HMEV and some dEV fractions contain mammary gland-derived protein BTN1A1. Neonatal human enteroids rapidly take up dEVs in part via clathrin-mediated endocytosis. Our data suggest that EVs can be isolated from digestive fluid and that these dEVs can be absorbed by IECs.
摘要：
人乳含有细胞外囊泡(HMEV)。临床前模型表明，HMEVs可能增强肠道功能并限制炎症；然而，尚不清楚HMEV或其货物是否在新生儿人类消化中存活。这限制了利用HMEV货物作为婴儿营养添加剂或作为治疗剂的能力。这项研究旨在从少量的人乳和乳后的新生儿肠内容物（消化物）中开发EV分离管道，以解决HMEV在体内新生儿消化中存活以吸收肠上皮细胞（IECs）的假设。Digesta是从鼻十二指肠采样管或造口术中收集的。在两步撇脂后，通过密度梯度超速离心从原料和巴氏灭菌的人乳和消化物中分离出EV，酪蛋白的酸沉淀，和多步骤过滤。电动汽车通过电子显微镜进行了验证，西方印迹,纳米粒子跟踪分析，电阻式脉冲传感,和超分辨率显微镜。在人新生儿肠样物质中测试了EV摄取。HMEV和消化型EV（dEV）显示典型的EV形态，富含CD81和CD9，但β-酪蛋白和乳白蛋白耗尽。HMEV和一些dEV部分含有乳腺衍生蛋白BTN1A1。新生儿类肠样物质部分通过网格蛋白介导的内吞作用快速吸收dEV。我们的数据表明，EV可以从消化液中分离出来，并且这些dEV可以被IEC吸收。
