Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Koç University Research Center for Translational Medicine (KUTTAM), Rumeli Fener, Sarıyer, Istanbul, Turkey.
Cell Tissue Res. 2021 May;384(2):389-401. doi: 10.1007/s00441-020-03405-4. Epub 2021 Jan 18.
Human endothelial progenitor cells (EPCs) were isolated from cord blood samples and enriched by magnetic activated cell sorting method based on the CD133 marker. Cells were incubated with different doses of bacterial lipopolysaccharide, ranging from 2, 5, 10, 50, 100, 200, 250, 500, to 1000 µg/ml, for 48 h. The cell survival rate was determined by using MTT assay. To confirm activation of the toll-like receptor signaling pathway, PCR array analysis was performed. Protein levels of ERK1/2, p-ERK1/2, NF-ƙB and TRIF proteins were measured using western blotting. The content of TNF-α and lipoprotein lipase activity were analyzed by immunofluorescence imaging. Flow cytometric analysis of CD31 was performed to assess the maturation rate. Cell migration was studied by the Transwell migration assay. The expression of genes related to exosome biogenesis was measured using real-time PCR analysis. In vivo gel plug angiogenesis assay was done in nude mice. Lipopolysaccharide changed endothelial progenitor cells' survival in a dose-dependent manner with maximum viable cells in groups treated with 2 µg/ml. PCR array analysis showed the activation of toll-like signaling pathways after exposure to LPS (p<0.05). Western blotting analysis indicated an induction of p-ERK1/2 and Erk1/2, NF-kB and TRIF in LPS-treated EPCs compared with the control (p<0.05). Immunofluorescence staining showed an elevation of TNF-α and lipoprotein lipase activity after lipopolysaccharide treatment (p<0.05). Lipopolysaccharide increased EPC migration and expression of exosome biogenesis-related genes (p<0.05). In vivo gel plug analysis revealed enhanced angiogenesis in cells exposed to bacterial lipopolysaccharide. Data highlighted the close relationship between the toll-like receptor signaling pathway and functional activity in EPCs.
人脐血内皮祖细胞（EPCs）通过基于 CD133 标记的磁激活细胞分选法分离并富集。将细胞用不同剂量的细菌脂多糖孵育，范围为 2、5、10、50、100、200、250、500 和 1000µg/ml，持续 48h。通过 MTT 测定法测定细胞存活率。为了确认 toll 样受体信号通路的激活，进行了 PCR 阵列分析。使用 Western blot 测定 ERK1/2、p-ERK1/2、NF-ƙB 和 TRIF 蛋白的蛋白水平。通过免疫荧光成像分析 TNF-α 和脂蛋白脂肪酶活性的含量。通过流式细胞术分析 CD31 评估成熟率。通过 Transwell 迁移测定研究细胞迁移。使用实时 PCR 分析测量与外泌体生物发生相关的基因的表达。在裸鼠体内进行凝胶塞血管生成测定。脂多糖以剂量依赖性方式改变内皮祖细胞的存活，用 2µg/ml 处理的组中存活的细胞最多。PCR 阵列分析显示 LPS 暴露后 toll 样信号通路的激活（p<0.05）。Western blot 分析表明与对照组相比，LPS 处理的 EPCs 中 p-ERK1/2 和 Erk1/2、NF-kB 和 TRIF 的诱导（p<0.05）。免疫荧光染色显示脂多糖处理后 TNF-α 和脂蛋白脂肪酶活性升高（p<0.05）。脂多糖增加 EPC 迁移和外泌体生物发生相关基因的表达（p<0.05）。体内凝胶塞分析显示暴露于细菌脂多糖的细胞中血管生成增强。数据强调了 toll 样受体信号通路与 EPC 功能活性之间的密切关系。
