| Objectives Overactivation of microglia and release of proinflammatory cytokines are mechanisms of radioactive brain injury.One of the first things we learn about is exosomes derived from human umbilical cord mesenchymal stem cells(hUC-MSCs-exo)has the ability of immune regulation.Observation of hUC-MSCs-exo affect BV2 proliferation and activation,explore the hUC-MSCs-exo on neural function and mechanism,the regulation of inflammatory reaction has provided the new mentality for the application of the treatment of radiation brain injury.Methods One of the first things we do is to extract human umbilical cord derived mesenchymal stem cells(hUC-MSCs)using improved enzymatic digestion method.Then we would purify cells and culture them.The hUC-MSCs-CM was treated by ultra-high speed centrifugation,hUC-MSCs-exo was isolated,extracted and purified,and the enriched transmembrane proteins CD9,CD81 and CD63 on the surface of exosomes were identified by Western blot(WB).Particle diameter and distribution were determined by Nanoparticle tracking analysis(NTA).hUC-MSCs-exo with concentrations of 0ug/ml,25 ug /ml,50 ug /ml,100 ug /ml and 200 ug /ml were set to stimulate BV2 cells,and CCK-8 method was used to measure cell viability at 24 h after stimulation.Microglia cells were stimulated by Lipopolysaccharide(LPS)to simulate the inflammatory environment of the nervous system.BV2 cells which under LPS stimulation was co-cultured with huc-mscs or huc-mscs-exo for 24 h,using enzyme-linked immunosorbent assay(ELISA)analysis proinflammatory factor IL-6,TNF-α and suppression of inflammatory factor IL-4,the content of IL-10;WB assay was used to detect the relative expression of Arginase-1(Arg-1).Results 1.The morphology of the hUC-MSCs was observed under an inverted phase contrast microscope: the cells were star-shaped at 24 h after isolation and extraction,and the cells after the second generation presented a uniform long spindle shape,similar to the morphology of fibroblasts.When the cell density grew to 80%-90%,the cells grew in parallel or spiral arrangement.2.The hUC-MSCs surface protein markers were detected by flow cytology.The results showed that the hUC-MSCs extracted and purified in this experiment were highly expressed in CD73-APC(99.9%),CD90-FITC(99.8%)and CD105-APC(98.5%).And no or low expression of HLA-DR-FITC(0.1%)CD45-FITC(0.1%)CD34-FITC(0.0%)CD11b-FITC(0.0%)and CD19-FITC(0.0%)3.WB technology was used to detect the protein markers of exosome.The results showed that the hUC-MSCs-exo extracted in this experiment was enriched with CD9,CD81 and CD63 transmembrane proteins.The diameter size and distribution of exosomes were detected by NTA,and the results showed that the diameter of most particles was 152nm(99.2%),suggesting that exosome particles with high purity could be obtained by overspeed centrifugation.4.At 24 h,different concentration of hUC-MSCs-exo can make BV2 cell viability decreased(P < 0.05),and exosomes concentration was negatively related to BV2 cell viability.5.Pure LPS stimulation group BV2 cells supernatant of proinflammatory factor secretion of IL-6 and TNF-α levels increased significantly(P<0.05),while it is giving after LPS stimulation at the same time give hUC-MSCs-exo or hUC-MSCs co-culture,these two kinds of cytokine secretion levels were not significantly higher(P>0.05).6.The secretion levels of anti-inflammatory factors IL-4 and IL-10 in the hUC-MSCs-exo group and the hUC-MSCs co-culture group were significantly increased(P<0.05),while the secretion levels of these two anti-inflammatory factors were not significantly increased in the LPS stimulation group(P>0.05).7.In the hUC-MSCs-exo and hUC-MSCs co-culture group,BV2 cells highly expressed the M2 microglial marker Arg-1(P<0.05).Conclusions 1.hUC-MSCs-exo can inhibit the proliferation activity of microglia cells,which is enhanced with the increase of exosome concentration.2.hUC-MSCs and hUCMSCs-exo can inhibit the secretion of microglia inflammatory factors IL-6 and TNF-α,and promote the secretion of anti-inflammatory factors IL-4 and IL-10.3.hUC-MSCs-exo can promote M2 activation of microglia.4.hUC-MSCs may achieve immunomodulatory effects by secreting exosomes.Figure 9;Table 4;Reference 108;... |
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