Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0.5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs), we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.
Objective To explore the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on the proliferation, migration, and tube-like differentiation of human umbilical vein endothelial cells (HUVECs). Methods Adipose tissue voluntarily donated by liposuction patients was obtained. The ADSCs were harvested by enzyme digestion and identified by flow cytometry and adipogenic induction. The ADSC-Exos were extracted from the supernatant of the 3rd generation ADSCs and the morphology was observed by transmission electron microscopy. The surface proteins (Alix and CD63) were detected by Western blot. The nanoparticle tracking analyzer NanoSight was used to analyze the size distribution of ADSC-Exos. After co-culture of PKH26 fluorescently labeled ADSC-Exos with HUVECs, confocal microscopy had been used to observe whether ADSC-Exos could absorbed by HUVECs. ADSC-Exos and HUVECs were co-cultured for 1, 2, 3, 4, and 5 days. The effect of ADSC-Exos on the proliferation of HUVECs was detected by cell counting kit 8 (CCK-8) assay. The expression of VEGF protein in the supernatant of HUVECs with or without ADSC-Exos had been detected by ELISA after 12 hours. Transwell migration assay was used to detect the effect of ADSC-Exos on the migration ability of HUVECs. The effect of ADSC-Exos on the tubular structure formation of HUVECs was observed by Matrigel experiments in vitro. The formation of subcutaneous tubular structure in vivo was observed in BALB/c male nude mice via the injection of HUVECs and Matrigel with or without ADSC-Exos. After 2 weeks, the neovascularization in Matrigel was measured and mean blood vessel density (MVD) was calculated. The above experiments were all controlled by the same amount of PBS. Results After identification, the cultured cells were consistent with the characteristics of ADSCs. ADSC-Exos were circular or elliptical membranous vesicle with uniform morphology under transmission electron microscopy, and expresses the signature proteins Alix and CD63 with particle size ranging from 30 to 200 nm. Confocal microscopy results showed that ADSC-Exos could be absorbed by HUVECs. The CCK-8 analysis showed that the cell proliferation of the experimental group was better than that of the control group at each time point (P<0.05). The result of Transwell showed that the trans-membrane migration cells in the experimental group were significantly more than that in the control group (t=9.534, P=0.000). In vitro, Matrigel tube-forming experiment showed that the number of tube-like structures in the experimental group was significantly higher than that of the control group (t=15.910, P=0.000). In vivo, the MVD of the experimental group was significantly higher than that of the control group (t=16.710, P=0.000). The ELISA assay showed that the expression of VEGF protein in the supernatant of the experimental group was significantly higher than that of the control group (t=21.470, P=0.000). Conclusion ADSC-Exos can promote proliferation, migration, and tube-like structure formation of HUVECs, suggesting that ADSC-Exos can promote angiogenesisin vitro and in vivo.
The aim of the study is to identify the effects and underlying mechanisms of visfatin on inflammation and necroptosis in vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were stimulated with visfatin or pretreated with Polyinosinic acid (LOX-1 inhibitor). By using the Western blot, RT-PCR, immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), MTT and flow cytometry technique, the occurrence of inflammation and necroptosis in HUVECs were evaluated. Our results showed that 100 ng/mL visfatin significantly increased the mRNA and protein expression of monocyte chemotactic protein 1 (MCP-1) and LOX-1 after 24 hours’ treatment in HUVECs. However, pretreatment with Polyinosinic acid could significantly reduce the expression of MCP-1 compared with visfatin group. Additionally, 100 ng/mL visfatin could induce the production of necrotic features and increase the mRNA expression of BMF (one of the markers of necroptosis), while pretreating with Polyinosinic acid markedly downregulated the mRNA expression of BMF gene and promoted the cell proliferation. These results indicate that visfatin might induce inflammation and necroptosis via LOX-1 in HUVECs, suggesting that visfatin plays a central role in the development of atherosclerosis.
ObjectiveTo investigate whether exosomes derived from miR-27a-overexpressing human umbilical vein endothelial cells (HUVECs)—exo (miR-27a) can promote bone regeneration and improve glucocorticoids (GC) induced osteonecrosis of femoral head (ONFH) (GC-ONFH).MethodsThe exo (miR-27a) were intended to be constructed and identified by transmission electron microscopy, nanoparticle tracking analysis, Western blot, and real-time fluorescent quantitative PCR (qRT-PCR). qRT-PCR was used to evaluate the effect of exo (miR-27a) in delivering miR-27a to osteoblasts (MC3T3-E1 cells). Alkaline phosphatase staining, alizarin red staining, and qRT-PCR were used to evaluate its effect on MC3T3-E1 cells osteogenesis. Dual-luciferase reporter (DLRTM) assay was used to verify whether miR-27a targeting Dickkopf WNT signaling pathway inhibitor 2 (DKK2) was a potential mechanism, and the mechanism was further verified by qRT-PCR, Western blot, and alizarin red staining in MC3T3-E1 cells. Finally, the protective effect of exo (miR-27a) on ONFH was verified by the GC-ONFH model in Sprague Dawley (SD) rats.ResultsTransmission electron microscopy, nanoparticle tracking analysis, Western blot, and qRT-PCR detection showed that exo (miR-27a) was successfully constructed. exo (miR-27a) could effectively deliver miR-27a to MC3T3-E1 cells and enhance their osteogenic capacity. The detection of DLRTM showed that miR-27a promoted bone formation by directly targeting DDK2. Micro-CT and HE staining results of animal experiments showed that tail vein injection of exo (miR-27a) improved the osteonecrosis of SD rat GC-ONFH model.Conclusionexo (miR-27a) can promote bone regeneration and protect against GC-ONFH to some extent.
Objective To investigate the effects of the MKN-45 gastric cancer cell exosomes carrying microRNA-552 (miR-552) on the proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVEC). Methods ① The MKN-45 cells were divided into MKN-45 blank control group (no transfection), MKN-45 miR-552 inhibitor group [transfection of plasmid inhibiting mir-552 expression (mir-552 inhibitor plasmid)], and MKN-45 negative control group [transfection of negative control plasmid (empty plasmid)], the exosomes were extracted, purified, and identified. Western blotting was used to detect the protein expression of exosomal markers [CD63, CD9, and tumor susceptibility gene 101 (TSG101)]. ② The HUVEC cells were divided into HUVEC control group (added PBS), HUVEC-exosome group (co-cultured with exosomes of MKN-45 cell), HUVEC-negative control exosome group (co-cultured with exosomes of MKN-45 cell transfected with negative control plasmid), and HUVEC-miR-552 inhibitor exosome group (co-cultured with exosomes of MKN-45 cell transfected with miR-552 inhibitor plasmid), exosomes tracing experiment was used to detect whether exosomes entered HUVEC cells. Real-time fluorescent quantitative PCR method was used to detect the expression of miR-552, the MTT method was used to detect the proliferation of HUVEC cells, the Transwell chamber method was used to detect the migration of HUVEC cells, the angiogenesis test was used to detect the angiogenesis ability. Results This study successfully extracted exosomes from MKN-45 gastric cancer cells. Observed by transmission electron microscope, the exosomes were all round or elliptical, with a diameter of 100–150 nm, and the exosomal vesicle structure could be seen. Western blotting detection showed that the surface markers of exosomes (CD63, CD9, and TSG101 protein) were expressed in exosomes. The results of the tracing experiment showed that exosomes derived from MKN-45 cells were successfully internalized by HUVEC cells. After MKN-45 cells were transfected with miR-552 inhibitor plasmid, compared with the MKN-45 blank control group and MKN-45 negative control group, the relative expression level of miR-552 in the exosomes decreased (P<0.05). Compared with the HUVEC control group, the cell proliferation rate at 24, 48 and 74 h increased, as well as number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-exosomes group increased (P<0.05). Compared with the HUVEC-negative control exosome group, the cell proliferation rate at 24, 48 and 74 h decreased, as well as the number of migration, tubule formation nodes, and relative expression level of miR-552 in the HUVEC-miR-552 inhibitor exosome group decreased (P<0.05). Conclusion The exosomes of gastric cancer cells carrying miR-552 can significantly promote the proliferation, migration, and angiogenesis of HUVEC cells.