Li Xiaoguo, Xu Mingjun, Ma Chen, Lyu Yuzhen, Ma Xiaona, Ma Xiaowei
Clinical Medical College, Ningxia Medical University, Yinchuan 750003; Ningxia Institute for Human Stem Cell Research, Yinchuan 750004; Intensive Care Unit, Cardiocerebral Vascular Disease Hospital, General Hospital of Ningxia Medical University, Yinchuan 750012, China.
Clinical Medical College, Ningxia Medical University, Yinchuan 750003; Ningxia Institute for Human Stem Cell Research, Yinchuan 750004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2021 Mar;37(3):225-232.
Objective To investigate the effect of exosomes derived from human placental mesenchymal stem cells (hPMSC-exs) on lipopolysaccharide (LPS)-induced injury of human pulmonary microvascular endothelial cells (HPMECs) and its possible mechanism. Methods hPMSCs were expanded and cultured in vitro and the cell culture supernatant was collected. The hPMSC-exs in the supernatant was separated and purified by ExoQuick exosomes extraction and purification kit. The morphological characteristics of exosomes were observed by transmission electron microscopy, and the expression of specific markers CD9 and CD63 on the surface of exosomes was detected by Western blotting. A non-contact co-culture system of hPMSCs and HPMECs was constructed. The experiment included a control group, an LPS injury group, an hPMSC group and an hPMSC-exs group. After 12 hours of co-cultivation, the fluorescence intensity of FITC-dextran from the upper chamber into the lower chamber was detected to reflect the permeability of single-layer pulmonary vascular endothelium; DiI-labeled hPMSC-exs was observed under a fluorescence microscope to judge the migration effect of hPMSC-exs on the damaged endothelium; CCK-8 assay was used to detect the proliferation level of the upper chamber cells in each group; JC-1 staining was used to detect the mitochondrial membrane potential of upper ventricular cells in each group; Western blotting was performed to detect the expression of autophagy-related protein LC3 and beclin-1 of HPMECs in the lower chamber. Results The diameter of hPMSC-exs was 30-120 nm, and the expression of CD9 and CD63 was positive, which matched the phenotypic characteristics of hPMSC-exs. Compared with the LPS injury group, the fluorescence intensity of FITC-dextran decreased; LC3II/I ratio and cell proliferation rate increased; beclin-1 protein expression was up-regulated in the hPMSC group and the hPMSC-exs group. Compared with the hPMSC group, the structure of endothelial cells was roughly intact under the fluorescence microscope; the FITC-dextran fluorescence intensity, endothelial cell proliferation rate, mitochondrial membrane potential, expression levels of LC3-II/I and beclin-1 did not change significantly in the hPMSC-exs group. Conclusion hPMSC-exs can alleviate the damage of HPMECs induced by LPS and improves mitochondrial function in the cells. Its mechanism may be related to enhance the autophagy of HPMECs.
目的 探讨人胎盘间充质干细胞来源的外泌体（hPMSC-exs）对脂多糖（LPS）诱导的人肺微血管内皮细胞（HPMECs）损伤的影响及其可能机制。方法 体外扩增培养人胎盘间充质干细胞（hPMSCs），收集细胞培养上清液。采用ExoQuick外泌体提取纯化试剂盒分离纯化上清液中的hPMSC-exs。通过透射电子显微镜观察外泌体的形态特征，采用蛋白质免疫印迹法检测外泌体表面特异性标志物CD9和CD63的表达。构建hPMSCs与HPMECs非接触共培养体系。实验分为对照组、LPS损伤组、hPMSC组和hPMSC-exs组。共培养12小时后，检测上室进入下室的FITC-葡聚糖荧光强度以反映单层肺血管内皮细胞的通透性；在荧光显微镜下观察DiI标记的hPMSC-exs，判断hPMSC-exs对受损内皮细胞的迁移作用；采用CCK-8法检测各组上室细胞的增殖水平；采用JC-1染色检测各组上室细胞的线粒体膜电位；采用蛋白质免疫印迹法检测下室HPMECs自噬相关蛋白LC3和beclin-1的表达。结果 hPMSC-exs直径为30～120 nm，CD9和CD63表达呈阳性，符合hPMSC-exs的表型特征。与LPS损伤组比较，FITC-葡聚糖荧光强度降低；LC3II/I比值和细胞增殖率升高；hPMSC组和hPMSC-exs组beclin-1蛋白表达上调。与hPMSC组比较，荧光显微镜下内皮细胞结构大致完整；hPMSC-exs组FITC-葡聚糖荧光强度、内皮细胞增殖率、线粒体膜电位、LC3-II/I和beclin-1表达水平无明显变化。结论 hPMSC-exs可减轻LPS诱导的HPMECs损伤，改善细胞线粒体功能。其机制可能与增强HPMECs自噬有关。
