Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA; Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine Perinatal Research, The University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX, 77555, USA.
Department of Obstetrics & Gynecology, Division of Maternal-Fetal Medicine Perinatal Research, The University of Texas Medical Branch at Galveston, 301 University Blvd., Galveston, TX, 77555, USA.
J Reprod Immunol. 2017 Sep;123:3-11. doi: 10.1016/j.jri.2017.08.003. Epub 2017 Aug 24.
Term labor in humans is associated with increased oxidative stress (OS) -induced senescence and damages to amnion epithelial cells (AECs). Senescent fetal cells release alarmin high-mobility group box 1 (HMGB1) and cell-free fetal telomere fragments (cffTF) which can be carried by exosomes to other uterine tissues to produce parturition-associated inflammatory changes. This study characterized AEC-derived exosomes under normal and OS conditions and their packaging of HMGB1 and cffTF. Primary AECs were treated with either standard media or oxidative stress-induced media (exposure to cigarette smoke extract for 48h). Senescence was determined, and exosomes were isolated and characterized. To colocalize HMGB1 and cffTF in amnion exosomes, immunofluorescent staining and in situ hybridization were performed, followed by confocal microscopy. Next generation sequencing (NGS) determined exosomal cffTF and other cell-free amnion cell DNA specificity. Regardless of condition, primary AECs produce exosomes with a classic size, shape, and markers. OS and senescence caused the translocation of HMGB1 and cffTF from AECs' nuclei to cytoplasm compared to untreated cells, which was inhibited by antioxidant N-acetyl cysteine (NAC). Linescans confirmed colocalization of HMGB1 and cffTF in exosomes were higher in the cytoplasm after CSE treatment compared to untreated AECs. NGS determined that besides cffTF, AEC exosomes also carry genomic and mitochondrial DNA, regardless of growth conditions. Sterile inflammatory markers HMGB1 and cffTF from senescent fetal cells are packaged inside exosomes. We postulate that this exosomal cargo can act as a fetal signal at term and can cause labor-associated changes in neighboring tissues.
人类的有期限劳动与氧化应激（OS）诱导的衰老和羊膜上皮细胞（AEC）损伤有关。衰老的胎儿细胞释放警报素高迁移率族蛋白 1（HMGB1）和无细胞胎儿端粒片段（cffTF），这些物质可以被外泌体携带到其他子宫组织中，产生与分娩相关的炎症变化。本研究对正常和 OS 条件下的 AEC 衍生外泌体及其 HMGB1 和 cffTF 的包装进行了特征描述。将原代 AEC 用标准培养基或氧化应激诱导培养基（暴露于香烟烟雾提取物 48 小时）处理。确定衰老情况，并分离和表征外泌体。为了在羊膜外泌体中对 HMGB1 和 cffTF 进行共定位，进行了免疫荧光染色和原位杂交，然后进行共聚焦显微镜检查。下一代测序（NGS）确定了外泌体 cffTF 和其他无细胞羊膜细胞 DNA 的特异性。无论条件如何，原代 AEC 都会产生具有经典大小、形状和标志物的外泌体。与未处理的细胞相比，OS 和衰老导致 HMGB1 和 cffTF 从 AEC 的核转移到细胞质，抗氧化剂 N-乙酰半胱氨酸（NAC）可抑制这种转移。线扫描证实，与未处理的 AEC 相比，CSE 处理后外泌体中 HMGB1 和 cffTF 的共定位在细胞质中更高。NGS 确定，除了 cffTF 之外，AEC 外泌体还携带基因组和线粒体 DNA，无论生长条件如何。来自衰老胎儿细胞的无菌炎症标志物 HMGB1 和 cffTF 被包装在 exosomes 中。我们推测，这种外泌体货物可以作为足月时的胎儿信号，并可以引起邻近组织与分娩相关的变化。
