Influences Of CXCL12 On Migration Ability, Stemness And Immunoregulation Effect Of Human Olfactory Mucosa Mesenchymal Stem Cells

ObjectiveTo investigate the influences of CXCL12 on migration ability,sternness and immunoregulation effect of human olfactory mucosa mesenchymal stem cells.Methods1.The culture and identification of human OM-MSCs:under the nasal endoscopy,a small piece of normal olfactory mucosa tissue with a diameter around 3mm were clipped apart from the hindside of the middle turbinate.Tissue adherent method was used to culture OM-MSCs.Cell surface markers involved were detected by flow cytometry,expressions of Nestin and STRO-1 were investigated by immunofluorescence.2.OM-MSCs were cultured till passage 3,then digested and passaged on 6 well plates.When the confluences of cells approached to 80%,cells were divided into 2 groups randomly,i.e.the negative control group and the experimental group.Cells in the negative control group were cultured with D/F-12 medium and 10%fetal bovine serum,while cells in the experimental group,additional recombinant protein CXCL12 at a concentration of 100ng/mL were added to the medium same with the negative control group.And then,cells of the two groups were cultured at 37?,CO2 at 5%,with saturated humidity in the incubator.3.A smooth linear scratch was made via a 10?L pipette tip on the cells of both groups,then cells were cultured in the incubator.The confluences of the scratches were captured after 24h and 48h via a microscope imaging system.4.Cultured after 48h,cells were collected and the total protein were extracted for Western Blotting of CXCR4(the receptor of CXCL12)and Nestin,in order to measure the differences in the expression levels of the two groups.5.Treated with CXCL12 after 48h,the supernatant of both groups were collected,stored and underwent the ELISA of IL-6.Results1.The morphological features of the cultured OM-MSCs were spindle-like or long and narrow strip-like.There could be several branches in one single cell,and cells grew in an adherent manner.When the confluence was high,cells grew in a vortex or parallel manner.The flow cytometry showed that the cultured OM-MSCs were CD34,CD45 negative and CD73,CD90,CD 105 positive.Immunoflurescence showed that OM-MSCs were Nestin and STRO-1 positive.Purity of cells was over 97%.These results indicate that the cultured OM-MSCs meets the standard of the identification of mesenchymal stem cells.2.In the scratch test,under the influence of CXCL12,the healing rate of the scratch in the experimental group was faster than the rate in the negative control group.3.In the Western Blotting of CXCR4,the expression in the experimental group was higher than in the negative control group,by gray scale analysis,P<0.01,it showed statistically significance.4.In the Western Blotting of Nestin,the expression in the experimental group was downregulated,by gray scale analysis,P<0.01,it showed statistically significance.5.In the ELISA test of secreted IL-6,the amount was markedly decreased in the experimental group,P<0.01,it showed statistically significance.Conclusion1.CXCL12 can enhance the migration ability of OM-MSCs directly,and also indirectly through upregulating the expression of CXCR4 in OM-MSCs.2.CXCL12 weakens the stemness of OM-MSCs via downregulating the expression of Nestin in OM-MSCs.3.CXCL12 changes the immunoregulation effects of OM-MSCs via downregulating the secreted IL-6 by OM-MSCs.