The Effect Of IFN-? On Proliferation And Immunosuppression In Canine BMSCs

Bone marrow mesenchymal stem cells(BMSCs)are multipotent adult stem cells.In addition to its strong ability of self-renewal and transdermal differentiation,BMSCs also have unique immunoregulation ability,which plays a role in inflammatory environment and can be directed to the site of injury of inflammatory tissue,and Interferon-?(IFN-?)is an important pro-inflammatory factor mediating various inflammatory injury.However,the mechanism of BMSCs immunomodulation is still unclear.The aim of this study was to investigate whether IFN-?can activate the immunosuppressive potential and participate immunomodulation of canine BMSCs,and explore the main methods and mechanisms of BMSCs.1.The effect of IFN-?on proliferation and secreting immune-related factors in canine BMSCs.Canine BMSC were treated with different concentrations of IFN-?after culturing72h.The proliferation were tested by CCK-8,the expression of immune-related genes were detected by RT-qPCR,and the secretion of immunosuppressive factors were tested by ELISA.The results were shown as follows:IFN-?can promote the proliferation of BMSCs with dose-dependent significantly,The expressions of TLR3 and IDO1 mRNA in BMSCs were up-regulated,the expressions of COX-2 was down-regulated(p<0.05),the expressions of TLR4 and IDO2 mRNA were not significantly different(p>0.05).The secretion of soluble immunosuppressive factors IL-10,HGF,and cyanine was increased,while the secretion of PGE2 and TGF-?1 was decreased(p<0.05).2.Effect of BMSCs and its supernatant on lymphocyte proliferation and immune function under IFN-?stimulation.Collecting and canine BMSCs and supernatant which treated with 50ng/ml IFN-?after culturing 72h,divided into the control group and the experimental groups.The control group(SPL)was cultured for lymphocyte.The six experimental groups including co-culture group(2):Unstimulated BMSCs group(BMSCs-nor group)and IFN-stimulated BMSCs group(BMSCs-IFN group)were co-cultured with 5?g/ml conA-activated lymphocytes at 1:10.Culture supernatant group(4):20%non-stimulating supernatant culture group(SI-nor group),40%non-stimulating supernatant culture group(SII-nor group),20%stimulating supernatant culture group(SI-IFN),40%stimulating supernatant culture group(SII-IFN)and RMPI-1640 were mixed-cultured with 5?g/ml conA-activated lymphocytes.The lymphocytes proliferation were tested by CCK-8,The CD3~+CD4~+CD8~+typing lymphocyte were observed by flow cytometry,the lymphocytes expression of Th1/Th2 subsets cytokines and proinflammatory factor mRNA were tested by RT-PCR.The results were shown as follows:Canine BMSCs can play an immunoregulatory role through direct cell contact or indirect culture with supernatant,including inhibiting lymphocyte proliferation,reducing CD4~+CD8~+content and its ratio,inducing lymphocyte differentiation into Th2 subgroups and inhibiting the expression of secreted inflammatory factor TNF-?,IL-8 mRNA.Conclusion:(1)This experiment proved that IFN-?can promote the proliferation of canine BMSCs,activated the expression of TLR3,IDO-1 mRNA,promoted the secretion of immunoactive factors,and enhanced the immunomodulatory effect of BMSCs on spleen lymphocytes in mice,mainly by inhibiting the proliferation of lymphocytes,reducing the Th2 subgroup differentiation of lymphocytes and inhibiting the expression of proinflammatory factor TNF-?mRNA.(2)The immunomodulatory effect of canine BMSCs on lymphocytes can be achieved by direct cell contact and indirect culture with supernatant,and the effect of direct cell contact on inhibiting lymphocyte proliferation and reducing CD4~+CD8~+content and its ratio is more significant.