Human Placental Mesenchymal Stem Cell Exosomes On Lipopolysaccharide-induced Pulmonary Vascular Endothelium Study Of Permeability Effects

Objective By observing the effect of human placental mesenchymal stem cell exosomes(hpMSC-EXOs)on lipopolysaccharide(LPS)-induced damage to monolayer human pulmonary capillary endothelial cells,we explored its effect on the permeability of monolayer capillary endothelial cells.Methods(1)(1)Human placental mesenchymal stem cells(hpMSCs)were expanded and cultured in vitro.(2)Identification of hpMSCs according to international definitions: Observe the growth morphology of hpMSCs under a light microscope,detect the expression of surface-specific antigens of human placental mesenchymal stem cells by flow cytometry,and induce differentiation of hpMSCs into the three lines of mesoderm cells(cartilage,osteogenesis,Adipogenic induction differentiation).(3)ExoQuick exosome extraction and purification reagent components were used to isolate and purify paracrine exosomes in the culture supernatant of human placental mesenchymal stem cells(hpMSC-EXOs).Western blot was used to detect hpMSC-EXOs surface specific antigen.The morphological characteristics of hpMSC-EXO were observed by transmission electron microscope.(2)(1)Detection of targeted metastases in exosomes:Co-culture of hpMSC-EXOs labeled with the Dil red fluorescent probe with normal HPMVEC and HPVEC induced by LPS was performed using a Transwell device,and the exosomes were transferred to HPMVEC under a fluorescence microscope.(2)Experiment grouping:Construct a co-culture system of hpMSCs,hpMSC-EXOs and human pulmonary microvascular endothelial cells(HPMVEC)Transwell,and set up a normal control group(NC group),lipopolysaccharide damage group(LPS group),and hpMSCs treatment group(hpMSCs + LPS group),hpMSC-EXOs treatment group(exosomes + LPS group).(3)Model preparation and treatment::Four groups of co-culture systems were inoculated with the same amount of HPMVEC.After the cells were completely adherent and 100% confluent,except for the normal control group,HPMVEC in the other three groups were intervened with 200 ng / mL lipopolysaccharide(LPS)for 6 h;After 6 hours,the lower chamber of the hpMSCs treatment group was inoculated with hpMSCs.The lower chamber of the hpMSC-EXOs treatment group was added with human placental mesenchymal stem cell exosomes.For 24 hours.(4)Detection of exosomes :After 24 hours,the exosomes were extracted and purified from the Transwell co-culture normal control group,lipopolysaccharide-damaged group,and hpMSCs treatment group in serum-free medium,and the presence of exosomes was identified by transmission electron microscopy.Western blot was used to detect exosome specific antigen.(5)Permeability measurement:FITC-dextran fluorescent protein permeability test was used to determine the permeability of human lung capillary endothelial monolayer in four groups of co-culture systems.Western blot experiments were performed to determine the expression levels of vascular endothelial-cadherin(VE-cadherin),claudin-1 and cytoskeleton protein(F-actin)in human pulmonary capillary endothelial tissue of each group.Results(1)(1)Human mesenchymal stem cells derived from human placental tissue were cultured for 24 hours in vitro,and they grew vortexly and adherently in a culture flask.Single cells were spindle-shaped and shaped like fibroblasts.(2)The expression of human placental mesenchymal stem cell surface antigens conforms to the typical MSC immunological phenotype: CD105,CD90,and CD73 are strongly positively expressed by flow cytometry;CD34,CD45,CD14,and HLA-DR are negatively expressed by flow cytometry.(3)human placental mesenchymal stem cells were induced into adipogenic,osteogenic,and chondrogenic culture 21 Days later,oil red O staining,alizarin red staining,and toluidine blue staining were all positive.(4)The morphology of hpMSC-EXOs were observed under the transmission electron microscope,which was cup-shaped or cake-shaped with a diameter between 30-100 nm.(5)hpMSC-EXOs expresses specific antigens CD9 and CD63.(2)(1)It were observed under fluorescent microscope that hpMSC-EXOs labeled with Dil red fluorescent probe can be transferred into normal human pulmonary vascular endothelial cells,but the red fluorescence expression in pulmonary vascular endothelial cells induced by LPS was more intense.(2)Exosomes were extracted from LPS group,hpMSCs + LPS group and NC group in serum-free medium after 24 hours of co-cultivation in Transwell.Only hpMSCs + LPS group could observe the presence of exosomes under the electron microscope and detect The expression of surface-specific antigens CD9 and CD63.(3)Compared with the NC group,the expression intensity of FITC-dextran in the LPS group was significantly increased(p <0.05);compared with the LPS group,the expression intensity of FITC-dextran in the hpMSCs + LPS group and the exosomes + LPS group was significantly reduced(p <0.05).(4)Compared with the NC group,the expression levels of VE-cadherin,claudin-1,and F-actin in pulmonary vascular endothelial cells in the LPS group were significantly reduced(p <0.05);compared with the LPS group,the hpMSCs + LPS group,exosomes + LPS The expression levels of VE-cadherin,claudin-1,and F-actin protein in pulmonary vascular endothelial cells in the group were increased(p <0.05).Conclusion Human placental mesenchymal stem cells can reduce LPS-induced damage to the monolayer pulmonary vascular endothelial permeability through paracrine exosomes.