摘要
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(HPMEC)损伤的影响及可能的机制。方法体外扩增培养hPMSC,收集细胞培养上清,ExoQuick外泌体提取和纯化试剂盒分离、纯化上清液中hPMSC-exs。透射电镜观察外泌体形态特征,Western blot法检测外泌体表面特异性标志物CD9、CD63的表达。构建hPMSC与HPMEC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 h后,检测各组上室渗透到下室中的异硫氰酸荧光素标记的葡聚糖(FITC-dextran)荧光强度以确定单层肺血管内皮的通透性;1,1′-十八烷基-3,3,3′,3′-四甲基吲哚羰花青高氯酸酯(DiI)标记hPMSC-exs并在荧光显微镜下观察对损伤内皮的迁移作用;CCK-8法检测各组上室细胞增殖水平;线粒体膜电位检测试剂盒检测各组上室内皮细胞线粒体膜电位;Western blot法检测下室HPMEC自噬相关蛋白微管相关蛋白1轻链3(LC3)、beclin-1的蛋白表达。结果获得的hPMSC-exs直径在30~120 nm,CD9、CD63表达阳性,符合hPMSC-exs表型特征;与LPS损伤组相比,hPMSC组及hPMSC-exs组FITC-dextran荧光强度值降低,LC3-Ⅱ/Ⅰ比值增加、beclin-1蛋白表达上调,细胞增殖率提高。与hPMSC组相比,hPMSC-exs组荧光显微镜下内皮细胞间连接大致完整、FITC-dextran荧光强度值、内皮细胞增殖率、线粒体膜电位、LC3-Ⅱ/Ⅰ比值及beclin-1表达水平均无明显变化。结论hPMSC-exs可减轻LPS诱导的HPMEC损伤,改善细胞内线粒体功能,其作用机制可能与增加HPMEC自噬有关。
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 upperchamber 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/Ⅰratio 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 intensi
作者
李晓国
徐明均
马晨
吕玉珍
马晓娜
马晓薇
LI Xiaoguo;XU Mingjun;MA Chen;LYU Yuzhen;MA Xiaona;MA Xiaowei(Clinical Medical Collge,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)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2021年第3期225-232,共8页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(81860350)
宁夏自然科学基金(2018A0705)
宁夏高等院校科学研究项目(NGY2017126)。
